Novel Compounds for Modulating Cell Proliferation

ABSTRACT

Novel styrylacrylonitrile compounds which are useful in treating a variety of cell proliferative disorders such as cancer are disclosed.

BACKGROUND OF THE INVENTION

A wide range of growth factors coordinate cell proliferation anddifferentiation. Malignant cells arise as a result of a stepwiseprogression of events that include the unregulated expression of growthfactors or components of their signaling pathways. Tyrosinephosphorylation events initiated by receptor, cytoplasmic and nuclearkinases and regulated by phosphatases are central to these processes.Mutation, hyper-activation, translocation and overexpression of proteintyrosine kinases are all associated with tumorigenesis. In addition toincreasing proliferative rates and immortalizing cells, overexpressionof tyrosine kinases can lead to morphological transformation and causeanchorage independence, contributing to the promotion of migratoryability and possibly the induction of metastases.

Certain compounds with structures based upon mimicry of ATP orphosphotyrosine have been shown to be effective kinase inhibitors. Thosebased upon phosphotyrosine have been demonstrated to be the morespecific tyrosine kinase inhibitors. Because of their ability to inhibittyrosine phosphorylation, these compounds may alter cell responses togrowth factors or other process driven by tyrosine kinase activity,including unregulated growth as the result of tyrosine kinaseoverexpression, mutation, or translocation. Inhibition of tyrosinekinases occupying a central role in proliferative signaling pathways, orin pathways regulating cell cytoskeletal structure, even temporary orincomplete inhibition, may be sufficient to switch a cancerous cell froma proliferative cycle into programmed cell death, or apoptosis. Death byapoptosis is most often observed upon effective treatment with tyrosinekinase inhibitors.

Selective inhibition of specific tyrosine kinases offers a method oftargeting cancerous cell growth with a high degree of specificity andminimal toxicity to normally growing cells and tissues. Thus, specificinhibitors of tyrosine kinases have great potential as clinicalanti-cancer treatments. A number of small molecules which act astyrosine kinase inhibitors have been identified. For example, certainphenyl acrylonitrile compounds have been described as tyrosine kinaseinhibitors, effective to inhibit cell proliferation (see for example,U.S. Pat. No. 5,891,917, U.S. Pat. No. 5,217,999, U.S. Pat. No.5,773,476, U.S. Pat. No. 5,935,993, U.S. Pat. No. 5,656,655, U.S. Pat.No. 5,677,329 and U.S. Pat. No. 5,789,427).

Inhibition of tyrosine kinases offers one mechanism by which cellproliferation can be inhibited. One of skill in the art will appreciatethat other mechanisms of inhibition may also be involved.

There is a need in the art to identify compounds that inhibit cellproliferation.

SUMMARY OF THE INVENTION

A number of novel compounds have now been identified that inhibitabnormal cell proliferation, for example cancer cell growth. Thecompounds do not inhibit the growth of normal cells.

Accordingly, the present invention includes a compound of Formula I or asalt, solvate, or hydrate thereof:

wherein

R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring;

R³ is selected from H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆alkyl(C═O)NH,C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

R⁴ is selected from C(X)R⁵, SO₃Ar, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), P(O)(OH)₂, P(O)(OC₁₋₆alkyl)₂, andC(NH₂)═C(CN)₂;

X is selected from O, S, NH and N—C₁₋₆alkyl;

R⁵ is selected from NH₂, OH, NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH,(CH₂)_(p)OC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkoxy, NHNH₂, NHC(O)NH₂,NHC(O)C₁₋₆alkoxy, N-morpholino and N-pyrrolidino;

Ar is an aromatic or heteroaromatic group, unsubstituted or substitutedwith 1-4 substituents, independently selected from OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,NO₂, CF₃, OCF₃ and halo;

n is 0 to 4; and

p is 1-4.

The present invention further includes a compound of Formula II or asalt, solvate, or hydrate thereof:

wherein

R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring;

R³ is selected from H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆alkyl(C═O)NH,C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

Ar is an aromatic or heteroaromatic group, unsubstituted or substitutedwith 1-4 substituents, independently selected from OH,

C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,

S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

R⁶ is selected from Ar, OH and OC₁₋₆alkyl;

X is selected from O and S;

n is 0-4; and

p is 1-4.

The present invention also provides a compound of Formula III or a salt,solvate, or hydrate thereof:

wherein

R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring;

R³ is selected from H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆alkyl(C═O)NH,C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

Ar is an aromatic or heteroaromatic group, unsubstituted or substitutedwith 1-4 substituents, independently selected from OH,

C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,

S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

R⁷ is selected from OH, NH₂ and OC₁₋₆alkyl;

X is selected from O and S; and

n is 0-4.

According to another aspect of the present invention, there is provideda pharmaceutical composition comprising a compound of the invention anda pharmaceutically acceptable carrier or diluent.

In accordance with a further aspect of the present invention, there isprovided a method for modulating cell proliferation, preferablyinhibiting cell proliferation comprising administering an effectiveamount of a compound of the invention to a cell or animal in needthereof. The invention also includes a use of a compound of theinvention to modulate cell proliferation, preferably inhibit cellproliferation. The invention further includes a use of a compound of theinvention to prepare a medicament to modulate cell proliferation,preferably inhibit cell proliferation.

In a preferred embodiment the present invention provides a method ofinhibiting the proliferation of a cancer cell comprising administeringan effective amount of a compound of the invention to a cell or animalin need thereof. The cancer cell treated may be any type of cancerincluding a leukemia, a lymphoma, myeloma, metastatic carcinoma, sarcomaor any other malignant transformation or any other malignancy. Theinvention also includes a use of a compound of the invention to modulatecancer cell proliferation, preferably inhibit cancer cell proliferation.The invention further includes a use of a compound of the invention toprepare a medicament to modulate cancer cell proliferation, preferablyinhibit cancer cell proliferation.

In another aspect, the invention provides a method of modulatingtyrosine kinase activity in a cell by administering an effective amountof a compound of the invention. In a further aspect, the inventionprovides a method of inhibiting tyrosine kinase activity in a cell byadministering an effective amount of a compound of the invention. Thepresent invention also provides a use of a compound of the invention tomodulate, preferably inhibit, tyrosine kinase activity. The presentinvention further provides a use of a compound of the invention toprepare a medicament to modulate tyrosine kinase activity, preferablyinhibit tyrosine kinase activity. It is appreciated that the inhibitionof cell growth by the compounds of the invention may be effected byother mechanisms.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is a bar graph showing the effect of CR4 upon normal bone marrowdifferentiation in culture.

FIG. 2 is a bar graph showing the killing of Philadelphia positive acutelymphoblastic leukemia by low-dose CR4 in culture.

FIG. 3 is a bar graph showing the killing of Philadelphia positive Z119Acute lymphoblastic leukemia cells by low-dose CR4 in culture.

FIG. 4 is a bar graph showing the killing of AML-3 acute myeloidleukemia cells by low-dose CR4 in culture.

FIG. 5 is a bar graph showing the killing of Ly-MN lymphoma cells bylow-dose CR4 in culture.

FIG. 6 is a bar graph showing the killing of primary juvenilemyelo-monocytic leukemia cells by CR4 in culture.

FIG. 7 is a bar graph showing the killing of OCI-LY2 lymphoma cells bylow-dose CR4 in culture.

FIG. 8 is a bar graph showing the killing of Philadelphia positive ALLcells by CR17 and CR21 in culture.

FIG. 9 is a bar graph showing the killing of Philadelphia positive ALLcells by CR17 and CR21 in culture.

FIG. 10 is a bar graph showing the killing of Philadelphia positive ALLcells by CR24 in culture.

FIG. 11 is a bar graph showing the killing of Philadelphia positive ALLcells by CR19 in culture.

FIG. 12 is bar graph showing the effect of CR19 on normal bone marrowdifferentiation in culture.

FIG. 13 is a bar graph showing the effect of CR24, CR17 and CR21 onnormal bone marrow differentiation.

FIG. 14 is a bar graph showing the effect of in vitro purging of normalbone marrow with CR4.

FIG. 15 is a bar graph showing the effect of in vitro purging of Z119acute lymphoblastic leukemia with CR4.

FIG. 16 is a bar graph showing the effect of in vitro purging of OCI-Ly2lymphoma cells with CR4.

FIG. 17 is a bar graph showing the effect of in vitro purging ofOCI-AML-3 acute meyloid leukemia cells with CR4.

FIG. 18 is a bar graph showing the effect of in vitro purging of Ramos Bcell Burkitt's lymphoma cells with CR4.

FIG. 19 is a bar graph showing the killing of HuNS1 multiple myelomacells by low-dose CR4 in culture.

FIGS. 20A and B are graphs showing cell staining after in vivo treatmentof Philadelphia positive acute lymphoblastic leukemia in NOD-SCID mice.

FIG. 21 is a bar graph showing that the effect of in vitro purging ofnormal bone marrow with CR11.

FIG. 22 is a bar graph showing that the effect of in vitro purging ofPhiladelphia positive acute lymphoblastic leukemia with CR11.

FIG. 23 is an autoradiograph which shows Philadelphia (Ph+) ALL linesZ119 and Z181 (5×10⁶ cells/point) immunoprecipitated with Bcr-Ablantibody.

FIG. 24 is an autoradiograph which shows Philadelphia (Ph+) ALL lineZ119 (5×10⁶ cells/point) immunoprecipitated with Jak2 antibody.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “C₁₋₆alkyl” as used herein means, unless otherwise stated,straight and/or branched chain alkyl radicals containing from one to sixcarbon atoms and includes methyl, ethyl, propyl, isopropyl, t-butyl andthe like.

The term “C₁₋₆alkoxy” as used herein means, unless otherwise stated,straight and/or branched chain alkoxy radicals containing from one tosix carbon atoms and includes methoxy, ethoxy, propyoxyl, isopropyloxy,t-butoxy and the like.

The term “C₁₋₄alkyl” as used herein means, unless otherwise stated,straight and/or branched chain alkyl radicals containing from one tofour carbon atoms and includes methyl, ethyl, propyl, isopropyl, t-butyland the like.

The term “C₁₋₄alkoxy” as used herein means, unless otherwise stated,straight and/or branched chain alkoxy radicals containing from one tofour carbon atoms and includes methoxy, ethoxy, propyoxyl, isopropyloxy,t-butoxy and the like.

The term “Ar” as used herein, means an unsubstituted or substituted aryland/or heteroaryl group which, in the case of heteroaryl, may contain upto two heteroatoms, wherein the constituents are independently selectedfrom OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo, andincludes unsubstituted or substituted phenyl, furyl, thienyl, indolyl,naphthyl, quinolyl and the like.

The term “halo” as used herein means halogen and includes chloro,fluoro, bromo, iodo and the like.

The term “pharmaceutically acceptable salt” means an acid addition saltor a basic addition salt which is suitable for or compatible with thetreatment of patients.

The term “compound of the invention” as used herein includes anycompound of the Formula I, II or III as defined herein (including allsalts, solvates or hydrates thereof) as well as any compound whosestructure is specifically depicted herein (including all salts, solvatesor hydrates thereof).

The term “pharmaceutically acceptable acid addition salt” as used hereinmeans any non-toxic organic or inorganic salt of any base compoundsrepresented by Formulae I, II and/or III or any of their intermediates.Illustrative inorganic acids which form suitable salts includehydrochloric, hydrobromic, sulfuric and phosphoric acids, as well asmetal salts such as sodium monohydrogen orthophosphate and potassiumhydrogen sulfate. Illustrative organic acids that form suitable saltsinclude mono-, di-, and tricarboxylic acids such as glycolic, lactic,pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric,ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids,as well as sulfonic acids such as p-toluene sulfonic and methanesulfonicacids. Either the mono or di-acid salts can be formed, and such saltsmay exist in either a hydrated, solvated or substantially anhydrousform. In general, the acid addition salts of compounds of Formulae I, IIand/or III are more soluble in water and various hydrophilic organicsolvents, and generally demonstrate higher melting points in comparisonto their free base forms. The selection of the appropriate salt will beknown to one skilled in the art. Other non-pharmaceutically acceptablesalts, e.g. oxalates, may be used, for example, in the isolation ofcompounds of Formulae I, II and/or III for laboratory use, or forsubsequent conversion to a pharmaceutically acceptable acid additionsalt.

The term “pharmaceutically acceptable basic addition salt” as usedherein means any non-toxic organic or inorganic base addition salt ofany acid compounds represented by Formulae I, II and/or III or any oftheir intermediates. Illustrative inorganic bases which form suitablesalts include lithium, sodium, potassium, calcium, magnesium or bariumhydroxide. Illustrative organic bases which form suitable salts includealiphatic, alicyclic or aromatic organic amines such as methylamine,trimethylamine and picoline or ammonia. The selection of the appropriatesalt will be known to a person skilled in the art.

The term “solvate” as used herein means a compound of Formulae I, IIand/or III, or a pharmaceutically acceptable salt of a compound ofFormulae I, II and/or III, wherein molecules of a suitable solvent areincorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents are ethanol, water and the like. When water is thesolvent, the molecule is referred to as a “hydrate”.

The term an “effective amount” or a “sufficient amount” of an agent asused herein is that amount sufficient to effect beneficial or desiredresults, including clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. For example, inthe context of administering an agent that inhibits cancer cellproliferation, an effective amount of an agent is, for example, anamount sufficient to achieve such a reduction in cancer cellproliferation as compared to the response obtained withoutadministration of the agent.

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results can include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminishment of extent of disease, stabilized (i.e. notworsening) state of disease, preventing spread of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

“Palliating” a disease or disorder means that the extent and/orundesirable clinical manifestations of a disorder or a disease state arelessened and/or time course of the progression is slowed or lengthened,as compared to not treating the disorder.

The term “modulate” as used herein includes the inhibition orsuppression of a function or activity (such as cell proliferation) aswell as the enhancement of a function or activity.

To “inhibit” or “suppress” or “reduce” a function or activity, such ascancer cell proliferation, is to reduce the function or activity whencompared to otherwise same conditions except for a condition orparameter of interest, or alternatively, as compared to anotherconditions.

The term “animal” as used herein includes all members of the animalkingdom including human. The animal is preferably a human.

The term “a cell” as used herein includes a plurality of cells.Administering a compound to a cell includes in vivo, ex vivo and invitro treatment.

The term “cancer cells” as used herein includes all forms of cancer orneoplastic disease.

II. Compounds of the Invention

Novel compounds which are useful in modulating cell proliferation wereprepared. As such the compounds are useful in treating cellproliferative diseases such as cancer.

Accordingly, the present invention provides a compound of Formula I, ora salt, solvate, or hydrate thereof:

wherein

R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring;

R³ is selected from H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆alkyl(C═O)NH,C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

R⁴ is selected from C(X)R⁵, SO₃Ar, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), P(O)(OH)₂, P(O)(OC₁₋₆alkyl)₂, andC(NH₂)═C(CN)₂;

X is selected from O, S, NH and N—C₁₋₆alkyl;

R⁵ is selected from NH₂, OH, NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH,(CH₂)_(p)OC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkoxy, NHNH₂, NHC(O)NH₂,NHC(O)C₁₋₆alkoxy, N-morpholino and N-pyrrolidino; and

Ar is an aromatic or heteroaromatic group, unsubstituted or substitutedwith 1-4 substituents independently selected from OH,

C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,

S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

n is 0 to 4;

m is 1 to 4; and

p is 1-4.

In embodiments of the invention, compounds of Formula I are those inwhich R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring. Inpreferred embodiments, R¹ and R² are each independently selected from H,OH, C₁₋₄alkyl, C₁₋₄alkoxy, C₁₋₄alkylCO₂, NH₂, NH—C₁₋₄alkyl,C₁₋₄alkyl(C═O)NH, C₁₋₄alkyl(C═O)N(C₁₋₄alkyl), SH, S—C₁₋₄alkyl,O—Si(C₁₋₄alkyl)(C₁₋₄alkyl)(C₁₋₄alkyl), NO₂, CF₃, OCF₃ and halo. In morepreferred embodiments, R¹ and R² are each independently selected from H,OH, OCH₃, CH₃CO₂, O—Si(CH₃)₂(^(t)Bu), S-Me, SH, CH₃CONH, CH₃CONCH₃, andNO₂. In the most preferred embodiment of the present invention R¹ and R²are both OH or OCH₃ or R¹ is OCH₃ and R² is OH.

In further embodiments of the present invention, the compounds ofFormula I include those in which R³ is selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar(where n is 0-4). In preferred embodiments, R³ is selected from H, OH,C₁₋₄alkyl, C₁₋₄alkoxy, C₁₋₄alkylCO₂, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), C₁₋₄alkyl(C═O)NH, C₁₋₄alkyl(C═O)N(C₁₋₄alkyl),SH, S—C₁₋₄alkyl, NO₂ and halo. In a more preferred embodiment, R³ isselected from H, OH, OCH₃, CH₃CO₂, SH, SMe, CH₃CONH, CH₃CONCH₃, NO₂ andhalo. In the most preferred embodiment, R³ is selected from H, OH andOCH₃.

In certain embodiments, R¹, R², and R³ are each independently selectedfrom H, C₁₋₄alkylCO₂, C₁₋₆alkyl(C═O)NH, and C₁₋₆alkyl(C═O)N(C₁₋₆alkyl),provided that at least one group is other than hydrogen.

Embodiments of the invention include compounds of Formula I wherein R⁴is selected from C(X)R⁵, SO₃Ar, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), P(O)(OH)₂, P(O)(OC₁₋₆alkyl)₂, and C(NH₂)═C(CN)₂(where m is 1-4). In preferred embodiments, R⁴ is selected from C(X)R⁵and C(NH₂)═C(CN)₂. More preferably, R⁴ is C(X)R⁵. When R⁴ is C(X)R⁵,embodiments of the invention include compounds where X is selected fromO, S, NH and N—C₁₋₆alkyl and R⁵ is selected from NH₂, OH, NH(CH₂)_(p)Ar,NH(CH₂)_(p)OH, (CH₂)_(p)OC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkoxy, NHNH₂,NHC(O)NH₂, NHC(O)C₁₋₆alkoxy, N-morpholino and N-pyrrolidino (where p is1-4). In preferred embodiments, X is O or S and R⁵ is selected from NH₂,OH, NH(CH₂)_(p)Ar, (CH₂)_(p)OH and C₁₋₄alkoxy, (where p is 1-3). Mostpreferred, are compounds of Formula I wherein X is O and R⁵ is selectedfrom NH₂, OH, NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH and OCH₃, (where p is 1-2).

The present invention includes compounds of Formula I wherein the term“Ar” means an unsubstituted or substituted aryl and/or heteroaryl groupwhich, in the case of heteroaryl, may contain up to two heteroatoms,wherein the optional substituents are independently selected from OH,C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo, and includes unsubstituted orsubstituted phenyl, furyl, thienyl, indolyl, naphthyl, quinolyl and thelike. In embodiments of the present invention, Ar is an unsubstitutedphenyl group or a phenyl group substituted with 1-4 substituentsoptionally selected from OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo. Inpreferred embodiments, Ar is an unsubstituted phenyl group or phenylgroup substituted with 1-2 substituents optionally selected from OH,C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl, N(C₁₋₄alkyl)(C₁₋₄alkyl), SH,S—C₁₋₄alkyl, NO₂, CF₃, OCF₃ and halo. In more preferred embodiments, Aris an unsubstituted phenyl group or phenyl group substituted with 1-2substituents optionally selected from OH, OCH₃, NH₂, NHCH₃, N(CH₃)₂, SH,SCH₃, CF₃, OCF₃ and halo. In the most preferred embodiment, Ar isselected from phenyl and 3,4-dihydroxyphenyl.

The present invention further includes a compound of Formula II or asalt, solvate, or hydrate thereof:

wherein

R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring;

R³ is selected from H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆alkyl(C═O)NH,C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

Ar is an aromatic or heteroaromatic group, unsubstituted or substitutedwith 1-4 substituents, independently selected from OH,

C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,

S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

R⁶ is selected from Ar, OH and OC₁₋₆alkyl;

X is selected from O and S;

n is 0-4; and

p is 1-4.

In embodiments of the invention, compounds of Formula II are those inwhich R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring. Inpreferred embodiments, R¹ and R² are each independently selected from H,OH, C₁₋₄alkyl, C₁₋₄alkoxy, C₁₋₄alkylCO₂, NH₂, NH—C₁₋₄alkyl,C₁₋₄alkyl(C═O)NH, C₁₋₄alkyl(C═O)N(C₁₋₄alkyl), SH, S—C₁₋₄alkyl,O—Si(C₁₋₄alkyl)(C₁₋₄alkyl)(C₁₋₄alkyl), NO₂, CF₃, OCF₃ and halo. In morepreferred embodiments, R¹ and R² are each independently selected from H,OH, OCH₃, CH₃CO₂, O—Si(CH₃)₂(^(t)Bu), S-Me, SH, CH₃CONH, CH₃CONCH₃, andNO₂. In the most preferred embodiment of the present invention R¹ and R²are both OH or OCH₃ or R¹ is OCH₃ and R² is OH.

In further embodiments of the present invention, the compounds ofFormula II include those in which R³ is selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂), Ar(where n is 0-4). In preferred embodiments, R³ is selected from H, OH,C₁₋₄alkyl, C₁₋₄alkoxy, C₁₋₄alkylCO₂, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), C₁₋₄alkyl(C═O)NH, C₁₋₄alkyl(C═O)N(C₁₋₄alkyl),SH, S—C₁₋₄alkyl, NO₂ and halo. In a more preferred embodiment, R³ isselected from H, OH, OCH₃, CH₃CO₂, SH, SMe, NO₂, CH₃CONH, CH₃CONCH₃, andhalo. In the most preferred embodiment, R³ is selected from H, OH andOCH₃.

In certain embodiments, R¹, R², and R³ are each independently selectedfrom H, C₁₋₄alkylCO₂, C₁₋₆alkyl(C═O)NH, and C₁₋₆alkyl(C═O)N(C₁₋₆alkyl),provided that at least one group is other than hydrogen.

The present invention further includes compounds of Formula II whereinthe term “Ar” means an unsubstituted or substituted aryl and heteroarylgroup which, in the case of heteroaryl, may contain up to twoheteroatoms, wherein the optional substituents are independentlyselected from OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo, andincludes unsubstituted or substituted phenyl, furyl, thienyl, indolyl,naphthyl, quinolyl and the like. In embodiments of the presentinvention, Ar is an unsubstituted phenyl group or a phenyl groupsubstituted with 1-4 substituents optionally selected from OH,C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo. In preferred embodiments, Ar is anunsubstituted phenyl group or phenyl group substituted with 1-2substituents optionally selected from OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂,NH—C₁₋₄alkyl, N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂, CF₃, OCF₃and halo. In more preferred embodiments, Ar is an unsubstituted phenylgroup or phenyl group substituted with 1-2 substituents optionallyselected from OH, OCH₃, NH₂, NHCH₃, N(CH₃)₂, SH, SCH₃, CF₃, OCF₃ andhalo. In the most preferred embodiment, Ar is selected from phenyl and3,4-dihydroxyphenyl.

The compounds of Formula II, include those in which R⁶ is selected fromAr, OH and OC₁₋₆alkyl and p is 1-4. In preferred embodiments, R⁶ isselected from Ar and OH and p is 1-2. Most preferably, when R⁶ is Ar, pis 1 and when R⁶ is OH, p is 2. Where R⁶ is Ar, Ar means anunsubstituted or substituted aryl and/or heteroaryl group which, in thecase of heteroaryl, may contain up to two heteroatoms, wherein theoptional substituents are independently selected from OH, C₁₋₆alkyl,C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,NO₂, CF₃, OCF₃ and halo, and includes unsubstituted or substitutedphenyl, furyl, thienyl, indolyl, naphthyl, quinolyl and the like. Inembodiments of the present invention, Ar is an unsubstituted phenylgroup or a phenyl group substituted with 1-4 substituents optionallyselected from OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo. Inpreferred embodiments, Ar is an unsubstituted phenyl group or phenylgroup substituted with 1-2 substituents optionally selected from OH,C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl, N(C₁₋₄alkyl)(C₁₋₄alkyl), SH,S—C₁₋₄alkyl, NO₂, CF₃, OCF₃ and halo. In more preferred embodiments, Aris an unsubstituted phenyl group or phenyl group substituted with 1-2substituents optionally selected from OH, OCH₃, NH₂, NHCH₃, N(CH₃)₂, SH,SCH₃, CF₃, OCF₃ and halo. In the most preferred embodiment, Ar isselected from phenyl and 3,4-dihydroxyphenyl.

Compounds of Formula II, further include those in which X is selectedfrom O and S. In preferred embodiments, X is O.

The present invention also provides a compound of Formula III or a salt,solvate, or hydrate thereof:

wherein

R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring;

R³ is selected from H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆alkyl(C═O)NH,C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂)_(n)Ar;

Ar is an aromatic or heteroaromatic group, unsubstituted or substitutedwith 1-4 substituents, independently selected from OH,

C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,

S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo;

R⁷ is selected from OH, NH₂ and OC₁₋₆alkyl;

X is selected from O and S; and

n is 0-4.

In embodiments of the invention, compounds of Formula III are those inwhich R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring. Inpreferred embodiments, R¹ and R² are each independently selected from H,OH, C₁₋₄alkyl, C₁₋₄alkoxy, C₁₋₄alkylCO₂, NH₂, NH—C₁₋₄alkyl,C₁₋₄alkyl(C═O)NH, C₁₋₄alkyl(C═O)N(C₁₋₄alkyl), SH, S—C₁₋₄alkyl,O—Si(C₁₋₄alkyl)(C₁₋₄alkyl)(C₁₋₄alkyl), NO₂, CF₃, OCF₃ and halo. In morepreferred embodiments, R¹ and R² are each independently selected from H,OH, OCH₃, CH₃CO₂, O—Si(CH₃)₂(^(t)Bu), S-Me, SH, CH₃CONH, CH₃CONCH₃, andNO₂. In the most preferred embodiment of the present invention R¹ and R²are both OH or OCH₃ or R¹ is OCH₃ and R² is OH.

In further embodiments of the present invention, the compounds ofFormula III include those in which R³ is selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂), Ar(where n is 0-4). In preferred embodiments, R³ is selected from H, OH,C₁₋₄alkyl, C₁₋₄alkoxy, C₁₋₄alkylCO₂, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), C₁₋₄alkyl(C═O)NH, C₁₋₄alkyl(C═O)N(C₁₋₄alkyl),SH, S—C₁₋₄alkyl, NO₂ and halo. In a more preferred embodiment, R³ isselected from H, OH, OCH₃, CH₃CO₂, SH, SMe, NO₂, CH₃CONH, CH₃CONCH₃, andhalo. In the most preferred embodiment, R³ is selected from H, OH andOCH₃.

In certain embodiments, R¹, R², and R³ are each independently selectedfrom H, C₁₋₄alkylCO₂, C₁₋₆alkyl(C═O)NH, and C₁₋₆alkyl(C═O)N(C₁₋₆alkyl),provided that at least one group is other than hydrogen.

The present invention further includes compounds of Formula III whereinthe term “Ar” means an unsubstituted or substituted aryl and/orheteroaryl group which, in the case of heteroaryl, may contain up to twoheteroatoms, wherein the optional substituents are independentlyselected from OH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo, andincludes unsubstituted or substituted phenyl, furyl, thienyl, indolyl,naphthyl, quinolyl and the like. In embodiments of the presentinvention, Ar is an unsubstituted phenyl group or a phenyl groupsubstituted with 1-4 substituents optionally selected from OH,C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH,S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo. In preferred embodiments, Ar is anunsubstituted phenyl group or phenyl group substituted with 1-2substituents optionally selected from OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂,NH—C₁₋₄alkyl, N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂, CF₃, OCF₃and halo. In more preferred embodiments, Ar is an unsubstituted phenylgroup or phenyl group substituted with 1-2 substituents optionallyselected from OH, OCH₃, NH₂, NHCH₃, N(CH₃)₂, SH, SCH₃, CF₃, OCF₃ andhalo. In the most preferred embodiment, Ar is selected from phenyl and3,4-dihydroxyphenyl.

Compounds of Formula III further include those in which R⁷ is selectedfrom OH, NH₂ and OC₁₋₆alkyl. In preferred embodiments, R⁷ is selectedfrom OH and NH₂.

Compounds of Formula III, further include those in which X is selectedfrom O and S. In preferred embodiments, X is O.

In specific embodiments of the present invention, the compounds of theinvention include:

-   (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (CR1);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile    (CR2);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR3);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR4);-   (E,E)-2-(phenylethylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile    (CR5);-   (E,E)-2-(phenylethylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR8);-   (E,E)-2-(phenylpropylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR9);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR11);-   (E,E)-2-thioacetaminocarbonyl-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR12);-   (E,E)-2-acetaminocarbonyl-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR13);-   (E,E)-2-carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR14);-   (E,E)-2-carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR15);-   (E,E)-2-acetaminocarbonyl-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile    (CR16);-   (E,E)-2-acetaminocarbonyl-3-(3,4-dihydroxystyryl)acrylonitrile    (CR17);-   (E,E)-2-(benzylaminocarbonyl)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile    (CR18);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-styrylacrylonitrile    (CR19);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile    (CR20);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR21);-   (E,E)-2-(β-ethanolaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR24);-   (E,E)-2-(benzylaminocarbonyl)-3-(4-nitrostyryl)acrylonitrile (CR27);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(4-nitrostyryl)acrylonitrile    (CR28); and-   (E,E)-2-(1-amino-2,2-dicyanoethenyl)-3-(4-nitrostyryl)acrylonitrile    (CR29).-   In preferred embodiments of the present invention, the compounds of    the invention include:-   (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (CR1);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile    (CR2);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR3);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR4);-   (E,E)-2-(phenylethylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile    (CR5);-   (E,E)-2-(phenylpropylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR9);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR11);-   (E,E)-2-thioacetaminocarbonyl-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR12);-   (E,E)-2-acetaminocarbonyl-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR13);-   (E,E)-2-carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR14);-   (E,E)-2-carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR15);-   (E,E)-2-acetaminocarbonyl-3-(3,4-dihydroxystyryl)acrylonitrile    (CR17);-   (E,E)-2-(3,4 dihydroxybenzylaminocarbonyl)-3-styrylacrylonitrile    (CR19);-   (E,E)-2-(3,4    dihydroxybenzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR21); and-   (E,E)-2-(β-ethanolaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR24).

In more preferred embodiments of the present invention, the compounds ofthe invention include:

-   (E,E)-2-(benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR4);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR11);-   (E,E)-2-acetaminocarbonyl-3-(3,4-dihydroxystyryl)acrylonitrile    (CR17);-   (E,E)-2-(3,4 dihydroxybenzylaminocarbonyl)-3-styrylacrylonitrile    (CR19);-   (E,E)-2-(3,4    dihydroxybenzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR21); and-   (E,E)-2-(β-ethanolaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR24).-   In certain embodiments of the present invention, the compounds of    the invention are selected from compounds having the structure of:

The present invention includes within its scope, prodrugs of thecompounds of the invention. In general, such prodrugs will be functionalderivatives of a compound of the invention which are readily convertiblein vivo into the compound from which it is notionally derived.Conventional procedures for the selection and preparation of suitableprodrugs are described, for example, in “Design of Prodrugs” ed. H.Bundgaard, Elsevier, 1985.

Some of the compounds of the invention may have at least one asymmetriccenter. Where the compounds according to the invention have oneasymmetric center, the may exist as enantiomers. Where the compounds ofthe invention possess two or more asymmetric centers, they mayadditionally exist as diastereomers. It is to be understood that allsuch isomers and mixtures thereof in any proportion are encompassedwithin the scope of the present invention.

The present invention includes radiolabeled forms of compounds of theinvention, for example, compounds of the invention labeled byincorporation within the structure ³H or ¹⁴C or a radioactive halogensuch as ¹²⁵I.

The compounds of the invention may, for example, be derived from anactivated cinnamyl compound and an activated cyano-substituted methylenecompound. A person skilled in the art, therefore, may wish to provide ageneric name for the compounds of the invention based on the cinnamylmoiety. However, generic nomenclature based on the formed acylonitrilemoiety, for example, styryl acrylonitrile, would be more proper.

III. Methods of Preparing Compounds of the Invention

In accordance with another aspect of the present invention, thecompounds of the invention can be prepared by processes analogous tothose established in the art. Therefore, compounds of this invention maybe prepared by the reaction sequence shown in Scheme 1:

Compounds of the general Formulae I, II and/or III useful in thepractice of this invention can be prepared by Knoevenagel condensationof α,β-unsaturated aldehydes, such as cinnamaldehyde or its variousaryl-substituted homologues (IV), with a compound having activeα-methylene group (V). Similar Knoevenagel condensations usingylidenemalononitriles as active α-methylene group components weredescribed in a review (F. Freeman. Chem. Rev. 1980, V. 80, P. 329-350).For example, these condensations may be carried out in a polar solvent,such as ethanol, in the presence of catalytic amounts of a weak base,such as β-alanine. Reaction temperatures may be in the range of 20 to100° C., depending on the stability of the materials used in thecondensation.

Compounds of Formulae IV and/or V may be commercially available, such ascinnamaldehyde, and its 3,5-dimethoxy-4-hydroxy derivative. Othercompounds of Formulae IV and/or V may be prepared using straightforwardprocedures. For example, various R¹, R², R³-hydroxy substitutedcinnamaldehydes can be prepared from the corresponding commerciallyavailable aryl substituted cinnamic acids. Scheme 2 gives an example ofthe preparation of protected 3,4-dihydroxycinnamaldehyde (IVa) startingfrom 3,4-dihydroxycinnamic acid (VI). At the end of the reactionsequence, the protection groups can be removed using standard methodswell known to those having skill in the art.

R¹, R², R³ substituents may be also converted from one functional groupto another, for example by known reduction of nitro groups into aminogroups and the further transformation into dialkylamino groups, or byknown conversion of hydroxy groups to halo groups.

α-Cyano amides with a reactive methylene group (Va) may be obtained, forexample, as described in A. Gazit et. al. J. Med. Chem., 1991, V. 34, P.1896-1907. For example, by heating methyl cyanoacetate (VII) and anappropriate commercially available amine (VIII) up to 100° C. withoutpresence of a solvent for 12-15 h followed by vacuum distillationdirectly from the mixture (for example using a Kugelrohr apparatus), thedesired products may be obtained (Scheme 3).

In some cases the chemistries outlined above may have to be modified,for instance by use of protective groups, to prevent side reactions dueto reactive groups, such as reactive groups attached as substituents.This may be achieved by means of conventional protecting groups, forexample as described in “Protective Groups in Organic Chemistry” McOmie,J. F. W. Ed., Plenum Press, 1973 and in Greene, T. W. and Wuts, P. G.M., “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1991.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

The formation of solvates of the compounds of the invention will varydepending on the compound and the solvate. In general, solvates areformed by dissolving the compound in the appropriate solvent andisolating the solvate by cooling or using an antisolvent. The solvate istypically dried or azeotroped under ambient conditions.

Prodrugs of the compounds of the invention may be conventional estersformed with available hydroxy, amino or carboxyl group. For example,when R¹, R² or R³ is OH in a compound of Formulae I, II and/or III, itmay be acylated using an activated acid in the presence of a base, andoptionally, in inert solvent (e.g. an acid chloride in pyridine). Somecommon esters which have been utilized as prodrugs are phenyl esters,aliphatic (C₈-C₂₄) esters, acyloxymethyl esters, carbamates and aminoacid esters.

A radiolabeled compound of the invention may be prepared using standardmethods known in the art. For example, tritium may be incorporated intoa compound of the invention using standard techniques, for example byhydrogenation of a suitable precursor to a compound of the inventionusing tritium gas and a catalyst. Alternatively, a compound of theinvention containing radioactive iodo may be prepared from thecorresponding trialkyltin (suitably trimethyltin) derivative usingstandard iodination conditions, such as [¹²⁵I] sodium iodide in thepresence of chloramine-T in a suitable solvent, such asdimethylformamide. The trialkyltin compound may be prepared from thecorresponding non-radioactive halo, suitably iodo, compound usingstandard palladium-catalyzed stannylation conditions, for examplehexamethylditin in the presence of tetrakis(triphenylphosphine)palladium (0) in an inert solvent, such as dioxane, and at elevatedtemperatures, suitably 50-100° C.

IV. Uses

As hereinbefore mentioned, the inventors have prepared novel compoundsof the Formulae I, II and III. Accordingly, the present inventionincludes all uses of the compounds of the invention including their usein therapeutic methods and compositions for modulating cellproliferation, their use in diagnostic assays and their use as researchtools.

In one aspect, the present invention provides a method for modulatingcell proliferation comprising administering an effective amount of acompound of the invention to a cell or animal in need thereof.Preferably, the invention provides a method of inhibiting cellproliferation comprising administering an effective amount of a compoundof the invention to a cell or animal in need thereof. In particular, themethod of the invention is useful in inhibiting the proliferation ofabnormal but not normal cells. Abnormal cells include any type of cellthat is causative of or involved in a disease or condition and whereinit is desirable to modulate or inhibit the proliferation of the abnormalcell to treat the disease or condition. Examples of abnormal cellsinclude malignant or cancerous cells as well as cell thatover-proliferate in inflammatory conditions.

It has been determined that some of the compounds of the invention arevery effective at killing cancer cells while at the same time they donot kill normal cells. These properties make the compounds of theinvention extremely useful as anti-cancer agents. Accordingly, in oneembodiment, the present invention provides a method of inhibiting theproliferation of a cancer cell comprising administering an effectiveamount of a compound of the invention to a cell or animal in needthereof.

The cancer cell that can be treated with a compound of the invention maybe any type of cancer including, but not limited to, hematopoieticmalignancies, including leukemias, lymphomas, and myelomas as well asother types of cancer including sarcomas, carcinomas, melanomas,adenomas, nervous system cancers and genitourinary cancers. Examples ofleukemias include acute lymphoblastic leukemia (ALL), acute myelocyticleukemia (AML), acute myelomonocytic leukemia (AMML), chronic myeloidleukemia (CML), chronic lymphocytic leukemia (CLL) and juvenilemyelo-monocytic leukemia (JMML). The types of ALL that may be treatedwith the compounds of the invention include cells that express a bcr-ablfusion protein, such as Philadelphia positive ALL cells, as well asPhiladelphia negative ALL cells. Examples of lymphomas include B-cellBurkitt's lymphoma, Hodgkin's lymphomas, non-Hodgkin's lymphomas,including the Ki-1 positive anaplastic large cell lymphomas, T celllymphomas and rare lymphomas such as the histiocytic lymphomas. Examplesof myelomas include multiple myelomas.

In a specific embodiment, the present invention provides a method ofinhibiting the proliferation of a cancer cell comprising administeringan effective amount of a compound selected from the group of compounds:

-   (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (CR1);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile    (CR2);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR3);-   (E,E)-2-(benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR4);-   (E,E)-2-(phenylethylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile    (CR5);-   (E,E)-2-(phenylethylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR8);-   (E,E)-2-(phenylpropylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR9);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR11);-   (E,E)-2-thioacetaminocarbonyl-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR12);-   (E,E)-2-acetaminocarbonyl-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR13);-   (E,E)-2-carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR14);-   (E,E)-2-carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR15);-   (E,E)-2-acetaminocarbonyl-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile    (CR16);-   (E,E)-2-acetaminocarbonyl-3-(3,4-dihydroxystyryl)acrylonitrile    (CR17);-   (E,E)-2-(benzylaminocarbonyl)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile    (CR18);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-styrylacrylonitrile    (CR19);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile    (CR20);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR21);-   (E,E)-2-(β-ethanolaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR24);-   (E,E)-2-(benzylaminocarbonyl)-3-(4-nitrostyryl)acrylonitrile (CR27);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(4-nitrostyryl)acrylonitrile    (CR28); and-   (E,E)-2-(1-amino-2,2-dicyanoethenyl)-3-(4-nitrostyryl)acrylonitrile    (CR29).-   In a preferred embodiment, the present invention provides a method    of inhibiting the proliferation of a cancer cell comprising    administering an effective amount of a compound selected from the    group of compounds:-   (E,E)-2-(benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR4);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR11);-   (E,E)-2-acetaminocarbonyl-3-(3,4-dihydroxystyryl)acrylonitrile    (CR17);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-styrylacrylonitrile    (CR19);-   (E,E)-2-(3,4-dihydroxybenzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile    (CR21); and-   (E,E)-2-(β-ethanolaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile    (CR24).-   In certain embodiments, the present invention provides a method of    inhibiting the proliferation of a cancer cell comprising    administering an effective amount of a compound selected from    compounds having the structures of:

One skilled in the art can determine which compounds of the inventionwould have therapeutic utility, for example, in inhibiting cellproliferation in any type of cancer or cell proliferative disorder.Compounds may be examined for their efficacy in inhibiting cell growthin cell proliferation assays such as those described herein in Examples35-56. Accordingly, the methods, uses and compositions of the inventionare meant to include only those compounds having the desired effect.

The ability of the compounds of the invention to inhibit the growth ofcancer cells, in particular hematopoetic cell malignancies, in vitro andin vivo was examined. Several of the compounds tested were found toeliminate cancerous cell growth in culture at sub-micro-molar doses. Inparticular, CR4, CR11 and CR19 were found to be highly effective againsta variety of cell types, such as Acute Lymphoblastic Leukemia,Philadelphia positive Leukemia and Acute Myeloid Leukemia. Low nanomolardoses of both CR4 and CR19 were highly toxic to cancer cells, whilenormal cell growth and differentiation were unaffected. These effectswere obtained by long term exposure to low levels of the compounds.Accordingly, in one aspect, this invention provides a method ofinhibiting the proliferation of a hematopoietic cancer cell byadministering an effective amount of a compound of the invention,preferably, CR4 or CR11 or CR19, to a cell or animal in need thereof.

It has been determined that the compound CR4 is capable of effectivelykilling human Philadelphia positive acute lymphoblastic leukemia cellsin vivo, using a murine model. CR4 efficiently reduced tumor load andinfiltration of the organs by the ALL cells. The doses required toeliminate cancer cell growth do not result in detectable non-specificdamage to the animal.

It has also been determined that the compounds of the invention, such asCR4 and CR11, are effective as ex vivo purging agents. For ex vivoadministration, bone marrow cells may be removed from a patient withcancer and purged ex vivo with a compound of the invention. Such apurging will kill the tumor cells while leaving the normal bone marrowcells intact. After purging, the cells can be washed and reintroducedinto the patient.

During ex vivo purging assays the cells were exposed to relatively highdoses of the compounds (50 μM-100 μM) for short (1-24 hours) periods oftime, resulting in the elimination of cancer cell growth, while normalbone marrow cells exposed to the same doses over the same period of timewere relatively unaffected. Cancer cell death was effected by theinduction of apoptosis. Accordingly, in another aspect of the invention,there is provided a method for killing cancer cells by ex vivo treatmentof bone marrow from a patient with cancer with a compound of theinvention, preferably CR4 and CR11 and then re-introducing the treated(or purged) bone marrow into the patient.

In addition to cancer, the compounds of the invention are useful intreating other conditions involving aberrant or abnormal cellproliferation. Other cell proliferative disorders that may be treated bythe present invention include inflammatory diseases, allergies,autoimmune disease, graft rejection psoriasis, restenosis,artherosclerosis, and any other disorder wherein it is desirable toinhibit, prevent or suppress cell growth. Compounds of the invention maybe tested for their efficacy in a particular cell proliferation disorderusing assays and techniques known to those of skill in the art. Forexample, the following references provide assays for various conditions.Rheumatoid Arthritis: “Regulation of IL-15—Simulated TNF-alphaProduction by Rolipram”, Journal of Immunology (1999) volume 163 page8236 by C. S. Kasyapa et al. Allergy: “A novel Lyn-Binding PeptideInhibitor Blocks Eosinophil Differentiation, Survival, and Airwayeosinophilic inflammation”. Journal of Immunology (1999) volume 163 page939 by T. Adachi et al. Psoriasis: Journal of Immunology (2000) volume165 page 224 “Inhibition of Keratinocyte apoptosis by IL-15: a newparamete in the pathegenosis of psoriasis” by R. Uchert (there is anumlart over the U). Psoriasis: International Archives of allergy andImmunology (2000) Volume 123 page 275. “T-cell receptor mimic peptidesand their potential application in T-cell mediated disease” by A. H.Enk.

The compounds of the invention are tyrosine kinase modulators and areuseful in modulating tyrosine kinase activity, including the inhibitionof tyrosine kinase activity, for the treatment of various conditionssuch as all proliferative disorders as mentioned above. Accordingly, theinvention provides a method of modulating tyrosine kinase activity byadministering an effective amount of a compound of the invention to acell or animal in need thereof. In a further aspect, the inventionprovides a method of inhibiting tyrosine kinase activity byadministering an effective amount of a compound of the invention to acell or animal in need thereof.

While the compounds of the invention may act by inhibiting tyrosinekinase activity, one of skill in the art will appreciate that othermodes or mechanisms of action for the compounds of the invention arepossible.

Certain of the subject compounds, including CR4,(E,E)-2-cyano-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile (CR7), CR8,CR11, CR19, and (E,E)2-(benzylaminocarbonyl)-3-(3-methoxy-4-hydroxystyryl)acrylonitrile(CR56) are capable of promoting myelopoiesis (defined herein asproliferation and/or differentiation of cells of the myeloid lineageincluding granulocytes, monocytes and its differentiated form,macrophages), in vitro, ex vivo and in vivo. See WO 03/030895, thecontent of which are incorporated herein by reference. Of the compoundsspecifically disclosed in this application, the following compounds alsopossess this activity:

Thus, the invention includes using any of these myelopoiesis-promotingcompounds in a method of promoting myelopoeisis in vivo, ex vivo, or invitro.

In one aspect, the invention relates to a method of promotingmyelopoiesis comprising administering an effective amount of one or moreof these myelopoiesis-promoting compounds to hematopoietic cell or ananimal in need thereof. The term “cell” includes a plurality of cells.Administration to a cell includes in vivo, ex vivo, and in vitrotreatment.

In some embodiments, the hematopoietic cell is hematopoeitic stem celland the animal is a human patient. In specific embodiments, thecompounds are administered to a human patient suffering from, or at riskof primary or secondary neutropenia, including chemotherapy or druginduced neutropenia, neutropenia secondary to malignancy, includingG-CSF responsive malignancies. In other embodiments, the compound(s) isadministered to a human patient at risk of, or suffering from aplasticanemia or aplasia. In other embodiments, the animal is a human donor ofbone marrow cells or peripheral blood stem cells. In other embodiments,one or more of these myelopoiesis-promoting compounds is administered toa human patient in need of bone marrow cell or peripheral blood stemcell transplant before or after the transplant.

In another aspect, the invention provides a method of promotingmyelopoiesis ex vivo comprising administering an effective amount of oneor more of these myelopoiesis-promoting compounds to a hematopoieticcell. In one embodiment, the cell is hematopoietic stem cell. Inspecific embodiments, the hernatopoietic cell is from the bone marrow orperipheral blood stem cells of a donor, or the bone marrow or peripheralblood stem cells of a patient in need of autologous bone marrow orperipheral blood stem cell transplant.

In another aspect, the invention provides a method of treating a patientsuffering from or at risk of neutropenia, aplastic anemia or aplasia,comprising administering an effective amount of one or more of thesemyelopoiesis-promoting compounds to said patient. In another aspect, theinvention provides a method of treating a patient suffering from or atrisk of neutropenia, aplastic anemia or aplasia, comprising introducinghematopoietic cells to the patient wherein one or more of thesemyelopoiesis-promoting compounds has been administered to the cells exvivo in an amount effective to promote myelopoiesis. The hematopoieticcells may be from the bone marrow or peripheral blood stem cells of adonor or of the patient.

In another aspect, the invention relates to use of one or more of thesemyelopoiesis-promoting compounds to promote myelopoiesis. The inventionalso relates to use of one or more of these myelopoiesis-promotingcompounds for preparing a medicament to promote myelopoiesis. Yet inanother aspect, the invention relates to use of one or more of thesemyelopoiesis-promoting compounds to treat neutropenia, aplastic anemiaor aplasia, and the use of one or more of these myelopoiesis-promotingcompounds to prepare a medicament to treat neutropenia aplastic anemiaor aplasia.

In another aspect, the invention provides a kit comprising one or moreof these myelopoiesis-promoting compounds and instructions for use,including to promote myelopoiesis and to treat neutropenia, aplasticanemia or aplasia.

The compounds of the invention are preferably formulated intopharmaceutical compositions for administration to human subjects in abiologically compatible form suitable for administration in vivo.Accordingly, in another aspect, the present invention provides apharmaceutical composition comprising a compound of the invention inadmixture with a suitable diluent or carrier.

The compositions containing the compounds of the invention can beprepared by known methods for the preparation of pharmaceuticallyacceptable compositions which can be administered to subjects, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle. Suitable vehicles aredescribed, for example, in Remington's Pharmaceutical Sciences(Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., USA 1985). On this basis, the compositions include, albeit notexclusively, solutions of the substances in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

The compounds of this invention may be used in the form of the freebase, in the form of salts, solvates and as hydrates. All forms arewithin the scope of the invention. Acid addition salts may be formed andprovide a more convenient form for use; in practice, use of the saltform inherently amounts to use of the base form. The acids which can beused to prepare the acid addition salts include preferably those whichproduce, when combined with the free base, pharmaceutically acceptablesalts, that is, salts whose anions are non-toxic to the animal organismin pharmaceutical doses of the salts, so that the beneficial propertiesinherent in the free base are not vitiated by side effects ascribable tothe anions. Although pharmaceutically acceptable salts of the basiccompounds are preferred, all acid addition salts are useful as sourcesof the free base form even if the particular salt per se is desired onlyas an intermediate product as, for example, when the salt is formed onlyfor the purposes of purification and identification, or when it is usedas an intermediate in preparing a pharmaceutically acceptable salt byion exchange procedures.

Pharmaceutically acceptable salts within the scope of the inventioninclude those derived from the following acids; mineral acids such ashydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; andorganic acids such as acetic acid, citric acid, lactic acid, tartaricacid, malonic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,quinic acid, and the like.

In accordance with the methods of the invention, the described compoundsor salts or solvates thereof may be administered to a patient in avariety of forms depending on the selected route of administration, aswill be understood by those skilled in the art. The compositions of theinvention may be administered orally or parenterally. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intramuscular, transepithelial, nasal, intrapulmonary, intrathecal,rectal and topical modes of administration. Parenteral administrationmay be by continuous infusion over a selected period of time.

A compound of the invention or a salt or solvate thereof may be orallyadministered, for example, with an inert diluent or with an assimilableedible carder, or it may be enclosed in hard or soft shell gelatincapsules, or it may be compressed into tablets, or it may beincorporated directly with the food of the diet. For oral therapeuticadministration, the compound of the invention may be incorporated withexcipient and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

A compound of the invention may also be administered parenterally orintraperitoneally. Solutions of a compound of the invention as a freebase or pharmacologically acceptable salt or solvate can be prepared inwater suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, DMSO and mixtures thereof with or without alcohol, and in oils.Under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms. A person skilledin the art would know how to prepare suitable formulations. Conventionalprocedures and ingredients for the selection and preparation of suitableformulations are described, for example, in Remington's PharmaceuticalSciences (1990—18th edition) and in The United States Pharmacopeia: TheNational Formulary (USP 24 NF19) published in 1999.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersion and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists.

The compounds of the invention may be administered to an animal alone orin combination with pharmaceutically acceptable carriers, as notedabove, the proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration andstandard pharmaceutical practice.

The dosage of the compounds and/or compositions of the invention canvary depending on many factors such as the pharmacodynamic properties ofthe compound, the mode of administration, the age, health and weight ofthe recipient, the nature and extent of the symptoms, the frequency ofthe treatment and the type of concurrent treatment, if any, and theclearance rate of the compound in the animal to be treated. One of skillin the art can determine the appropriate dosage based on the abovefactors. The compounds of the invention may be administered initially ina suitable dosage that may be adjusted as required, depending on theclinical response. As an example, the compounds of the invention can beadministered in a range from about 1 nanomolar to about 100 micromolar,preferably 50 nanomolar to 50 micromolar. For ex vivo treatment of cellsover a short period, for example for 30 minutes to 1 hour or longer,higher doses of compound may be used than for long term in vivo therapy;for example, concentrations of 50 μM or higher may be used.

The present invention also includes a use of a compound or compositionof the invention in order to inhibit cell proliferation, preferablycancer cell proliferation. The present invention further includes a useof a compound or a composition of the invention to prepare a medicamentto inhibit cell proliferation, preferably cancer cell proliferation.

The compounds of the invention can be used alone or in combination withother agents that modulate tyrosine kinase activity or in combinationwith other types of treatment (which may or may not modulate tyrosinekinase activity) for cell proliferative disorders. Agents known in theart that inhibit tyrosine kinase activity include, but are not limitedto, antisense nucleic acid and ribozymes targeted to nucleic acidencoding a receptor tyrosine kinase, antibodies able to modulatetyrosine kinase activity and other small molecule tyrosine kinaseinhibitors such as those described in U.S. Pat. No. 5,891,917, U.S. Pat.No. 5,217,999, U.S. Pat. No. 5,773,476, U.S. Pat. No. 5,935,993, U.S.Pat. No. 5,656,655, U.S. Pat. No. 5,677,329 and U.S. Pat. No. 5,789,427.There are various examples of other types of treatment for cellproliferative disorders currently used to treat different types ofcancers. The general treatments are based on the cancer type and do notspecifically target tyrosine kinase activity. In a particular aspect ofthe present invention, the compounds of the invention may be used incombination with other therapies and therapeutics to treat leukemia.

In addition to the above-mentioned therapeutic uses, the compounds ofthe invention are also useful in diagnostic assays, screening assays andas research tools.

In diagnostic assays the compounds of the invention may be useful inidentifying or detecting a cell proliferative disorder. In such anembodiment, the compounds of the invention may be radiolabelled (ashereinbefore described) and contacted with a population of cells. Thepresence of the radiolabelled on the cells may indicate a cellproliferative disorder. In a specific embodiment, the radiolabelledcompounds of the invention may be used to detect the presence of cellsexpressing a bcr-abl fusion protein.

In screening assays, the compounds of the invention may be used toidentify other compounds that modulate cell proliferation or tyrosinekinase activity. As research tools, the compounds of the invention maybe used in receptor binding assays and assays to study the localizationof tyrosine kinases. In such assays, the compounds may also beradiolabelled.

The following non-limiting examples are illustrative of the presentinvention:

EXEMPLIFICATION Materials and Methods for Examples 1-34

¹H NMR spectra were obtained on a Varian Unity Plus spectrometer (USA)at 500 MHz with tetramethylsilane (TMS, Me₄Si) as an internal standard(δ=0). Electrospray mass spectra were recorded on an API III Plus triplequadrupole mass spectrometer (USA), with a direct introduction of thesamples into the ionization source. Thin layer chromatography wasperformed with UV-254 aluminum-backed TLC sheets of 0.25 mm thickness(Kieselgel 60 F₂₅₄, Merck, Germany). HPLC separation of the compound ofExample 13 was performed on a Waters 600 chromatograph (USA), columnNova-Pak C18 3.9×300 mm (Waters, USA). Vacuum distillations were doneusing Kugelrohr apparatus (Aldrich, USA) at stated temperatures of anoven.

3,5-Dimethoxy-4-hydroxycinnamaldehyde, 4-nitrocinnamaldehyde,3,4-dimethoxycinnamic acid, 3,4-dihydroxycinnamic acid,3,4-dimethoxybenzylamine, benzylamine, phenylethylamine,phenylpropylamine, methyl cyanoacetate, 2-cyanothioacetamide,2-cyanoacetamide, cyanoacetic acid, β-ethanolamine,2-amino-1-propene-1,1,3-tricarbonitrile were purchased from Aldrich(USA) and were used as received. The reagents were from Aldrich (USA).Solvents were purchased from Calcdon (Canada).

Example 1 N-(Cyanoacetyl)3,4-dimethoxybenzylamide (A₁)

To 3,4-dimethoxybenzylamine (2.7 ml, 18 mmol) methyl cyanoacetate wasadded (1.6 ml, 18 mmol). The reaction was heated for 14 h at 100° C.Cooling gave a dark brown solid which was recrystallized from ethanol togive 2.90 g of the product (69% yield).

The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 3.62 (s, 2H, CH₂CN), 3.78 (s, 6H, (OMe)₂), 4.34(br.s., 2H, NHCH₂Ph), 6.84 (dd, 1H, J 1.95 and 8.1 Hz, H⁶), 6.88 (d, 1H,J=8.1 Hz, H⁵), 6.93 (d, 1H, J=1.95 Hz, H²), 7.80 (br.s., 1H, NH).

MS, m/e (rel. intensity, %): 235 (19) [M+H]⁺, 252 (100) [M+NH₄]⁺, 257(33) [M+Na]⁺.

Example 2 N-(Cyanoacetyl)3,4-dihydroxybenzylamide (A₂)

To N-(cyanoacetyl)3,4-dimethoxybenzylamide (Example 1, 0.2 g, 0.85 mmol)in 20 ml of CH₂Cl₂ boron tribromide was added under argon at −78° C.(0.24 ml, 2.56 mmol) in 2.5 ml of CH₂Cl₂. After 2 h the reaction wasbrought to room temperature and stirred overnight. The reaction wascooled to 0° C., 10 ml of 1N HCl was added, the solution was extractedwith 3×50 ml of ethyl acetate, the organic phase washed to neutral pH,dried with MgSO₄, and taken to dryness. The residue was purified bysilica gel chromatography (CHCl₃-MeOH, 20:1) to give a yellow solid(0.07 g, 40% yield). The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 2.83 (s, (OH)₂), 3.60 (s, 2H, CH₂CN), 4.25(br.s., 2H, NHCH₂Ph), 6.63 (dd, 1H, J 1.95 and 8.1 Hz, H⁶), 6.75 (d, 1H,J=8.1 Hz, H⁵), 6.79 (d, 1H, J=1.95 Hz, H²), 7.71 (br.s., 1H, NH).

MS, m/e (rel. intensity, %): 207 (38) [M+H]⁺, 224 (100) [M+NH₄]⁺, 229(2.6) [M+Na]⁺.

Example 3(E,E)-2-(3,4-Dihydroxybenzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR11)

To 3,5-dimethoxy-4-hydroxycinnamaldehyde (0.042 g, 0.2 mmol) andN-(cyanoacetyl)3,4-dihydroxybenzylamide (Example 2, 0.042 g, 0.2 mmol)in 10 ml of ethanol 3 mg of β-alanine was added and the reaction wasrefluxed for 6 h. Water was added and the solid was recrystallized from5 ml of ethanol twice to give 0.06 g (75%) of a red solid. The productgave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 2.81 (s, (OH)₃), 3.89 (s, 6H, (OMe)₂), 4.39(br.s., 2H, NHCH₂Ph), 6.68 (dd, 1H, J 1.95 and 8.1 Hz, H^(6′)), 6.76 (d,1H, J=8.1 Hz, H^(5′)), 6.86 (d, 1H, J=1.95 Hz, H^(2′)), 7.07 (br.s, 2H,H²⁺⁶), 7.16 (dd, 1H, J 11.7 and 15.1 Hz, PhCCHCCN olefinic), 7.37 (d,1H, J=15.1 Hz, PhCH olefinic), 7.70 (br.s., 1H, NH), 7.98 (dd, 1H, J0.75 and 11.7 Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 397 (100) [M+H]⁺, 414 (14) [M+NH₄]⁺.

Example 4 N-(Cyanoacetyl)benzylamide (A₃)

The compound was prepared as described in Example 1 by adding methylcyanoacetate (1.3 ml, 14 mmol) to benzylamine (1.5 ml, 14 mmol). Thecompound was distilled in vacuo directly from the reaction mixture(Kugelrohr apparatus (Aldrich), 0.1 mm Hg, T. oven 180-190° C.) to givean off-white solid (2.34 g, 95%). The product gave the followinganalytical data:

NMR (CD₃COCD₃, δ, ppm): 3.39 (s, 2H, CNCH₂), 4.46 (d, 2H, J=5.4 Hz,NHCH₂Ph), 6.40 (br.s., 1H, NH), 7.24-7.36 (m, 5H, Ph).

MS, m/e (rel. intensity, %): 175 (64) [M+H]⁺, 192 [M+NH₄]⁺.

Example 5 3,4-Dimethoxycinnamyl Alcohol (A₆)

To a solution of 0.42 g (2.0 mmol) of 3,4-dimethoxycinnamic acid in 50ml MeOH was added SOCl₂ (50 μl) and the mixture was stirred at 60° C.for 5 h. Methanol was taken to dryness and the obtained3,4-dimethoxycinnamic acid methyl ester was reduced with 1M THF solutionof diisobutylaluminum hydride (8.0 mmol) in absolute THF (50 ml) at 20°C. for 1 h. Water was added, the mixture was extracted with EtOAc, driedwith MgSO₄ and distilled in vacuo (Kugelrohr apparatus (Aldrich), 0.1 mmHg, T. oven 185-190° C.) giving an off-white solid, yield 0.36 g (92%),m.p. 70-71° C. The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 3.77, 3.82 (2×s, 2×3H, OMe+OMe), 4.19 (d, 2H,J=5.0 Hz, CH₂OH), 6.25 (dt, 1H, J 5.0 and 15.5 Hz, PhCCH olefinic), 6.51(d, 1H, J=15.5 Hz, PhCH olefinic), 6.89 (m, 2H, H⁵⁺⁶), 7.05 (br.s., 1H,H²).

MS, m/e (rel. intensity, %): 177 (100) [M-OH]⁺, 195 (4) [M+H]⁺, 212 (59)[M+NH₄]⁺, 217 (26) [M+Na]⁺.

Example 6 3,4-Dimethoxycinnamaldehyde (A₇)

To a mixture of pyridinium dichromate (3.88 g, 10.3 mmol) and 4 g offinely grounded freshly activated molecular sieves 3 Å in 20 ml ofCH₂Cl₂ 3,4-dimethoxycinnamyl alcohol in 10 ml of CH₂Cl₂ (Example 5, 1.00g, 5.1 mmol) was added. The reaction was stirred for 2 h, 0.5 ml ofmethanol was added, the residue was passed through silica gel and washedwith 300 ml of ethyl acetate. After evaporation the compound waspurified by silica gel chromatography (hexane-EtOAc, 5:1) leading to acrystallizing oil (0.62 g, 63%).

The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 3.90 (2×s, 2×3H, OCH₃+OCH₃), 6.70 (dd, 1H, J 7.6and 16.0 Hz, PhC═CH olefinic), 7.05 (d, 1H, J=8.3 Hz, H⁵), 7.28 (dd, 1H,J 1.4 and 8.3 Hz, H⁶), 7.37 (d, 1H, J=1.4 Hz, H²), 7.60 (d, 1H, J=16.0Hz, PhCH olefinic), 9.65 (d, 1H, J=7.6 Hz, CHO).

MS, m/e (rel. intensity, %): 193 (100) [M+H]⁺, 210 (26) [M+NH₄]⁺.

Example 7(E,E)-2-(Benzylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile (CR2)

The compound was prepared as described in Example 3, by adding3,4-dimethoxycinnamaldehyde (Example 6, 0.04 g, 0.2 mmol) toN-(cyanoacetyl)benzylamide (Example 4, 0.036 g, 0.2 mmol). Afterrefluxing for 1 h and recrystallization from ethanol a yellow solid wasobtained (0.045 g, 62%). The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 3.90 (s, 2×3H, OMe+OMe), 4.57 (d, 2H, J<2 Hz,NHCH₂Ph), 7.08 (br.s., 1H, H²), 7.17 (dd, 1H, J 1.5 and 15.2 Hz,PhCCHCCN olefinic), 7.23-7.42 (m, 8H, aromatic+H⁵+H⁶+PBCH olefinic),7.90 (br.t, 1H, NH), 8.05 (dd, 1H, J 0.55 and 11.5 Hz, CHCN olefinic).

Example 8(E,E)-2-(Benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile(CR4)—Method A

Boron tribromide (0.033 ml, 0.34 mmol) was added to(E,E)-2-(benzylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile(Example 7, 0.04 g, 0.11 mmol). The residue was purified by silica gelchromatography (CHCl₃-MeOH, 10:1) to give an orange solid (0.02 g, 55%yield). The product gave the following analytical data:

NMR (CD₃COCD₃, 8, ppm): 2.86 (br.s., 2H, (OH)₂), 4.55 (m, 2H, NHCH₂Ph),6.90-7.42 (m, 10H, Ph+Ph′+olefinic), 7.87 (br.s., 1H, NH), 8.02 (dd, 1H,J<0.5 and 11.4 Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 295 (61) [M+H—CN]⁺, 321 (100) [M+H]⁺, 338(30) [M+NH₄]⁺.

Example 9 Methyl ester of 3,4-bis(t-butyldimethylsilyloxy)cinnamic acid(A₈)

To a solution of 3.6 g (20 mmol) of 3,4-dihydroxycinnamic acid in 300 mlMeOH was added SOCl₂ (100 μl) and the mixture was stirred at 60° C. for5 h. Methanol was taken to dryness and the obtained methyl ester wastreated up with 10.2 g (68 mmol) of t-BuMe₂SiCl and 9.2 g (136 mmol) ofimidazole in 100 ml DMF at 50° C. for 0.5 h. Mixture was diluted withwater and extracted with hexane. Hexane was taken to dryness. Theresidue was distilled in vacuo (Kugelrohr apparatus (Aldrich), 0.1 mmHg, T. oven 200-210° C.) and crystallized from hexane at −20° C. givinga white solid, yield 7.5 g (89%), m.p. 57-58° C. The product gave thefollowing analytical data:

MS, m/e (rel. intensity, %): 423 (100) [M+H]⁺, 440 (98) [M+NH₄]⁺.

Example 10 3,4-Bis(t-butyldimethylsilyloxy)cinnamyl Alcohol (A₉)

The compound was prepared as described in Example 5 by treating of3,4-dihydroxycinnamic acid bis(BDMS) ether methyl ester (Example 9, 0.42g, 10.0 mmol) with 1M THF solution of diisobutylaluminum hydride (4.0mmol) in absolute THF (25 ml) at 20° C. for 1 h. After distilling invacuo (Kugelrohr apparatus (Aldrich), 0.1 mm Hg, T. oven 185-200° C.) awhite viscous oil was obtained, yield 0.33 g (85%). The product gave thefollowing analytical data:

NMR (CD₃COCD₃, δ, ppm): 0.23, 0.24 (2×s, 2×6H, Me₂Si+Me₂Si), 1.00, 1.02(2×s, 2×9H, t-BuSi+t-BuSi), 4.19 (d, 2H, J=4.9 Hz, CH₂OH), 6.22 (dt, 1H,J 4.9 and 16.0 Hz, PhCCH olefinic), 6.49 (d, 1H, J=16.0 Hz, PhCHolefinic), 6.85 (d, 1H, J=8.2 Hz, H⁵), 6.92 (dd, 1H, J 2.1 and 8.2 Hz,H⁶), 6.97 (d, 1H, J=2.1 Hz, H²).

MS, m/e (rel. intensity, %): 377 (100) [M-OH]⁺, 395 (2) [M+H]⁺, 412 (15)[M+NH₄]⁺.

Example 11 3,4-Bis(t-butyldimethylsilyloxy)cinnamaldehyde (A₁₀)

The compound was prepared as described in Example 6 by adding3,4-bis(t-butyldimethylsilyloxy)cinnamyl alcohol (Example 10, 0.2 g, 0.5mmol) in 5 ml of CH₂Cl₂ to a mixture of pyridinium dichromate (0.38 g, 1mmol) and 1 g molecular sieves 3 Å in 20 ml of CH₂Cl₂. The residue waspassed through silica gel and washed with 300 ml of EtOAc-hexane, 1:1.After evaporation the compound was purified by silica gel chromatography(hexane-EtOAc, 5:1) leading to an oil (0.15 g, 76%). The product gavethe following analytical data:

NMR (CD₃COCD₃, δ, ppm): 0.26 and 0.28 (2×s, 2×6H, Me₂Si+Me₂Si), 1.01 and1.02 (2×s, 2×9H, t-BuSi+t-BuSi), 6.60 (dd, 1H, J 7.7 and 15.9 Hz, PhCCHolefinic), 7.01 (dd, 1H, J<0.5 and 8.9 Hz, H⁶), 7.27 (m, 2H, H²⁺⁵), 7.60(d, 1H, J=15.9 Hz, PhCH olefinic), 9.65 (d, 1H, J=7.7 Hz, CHO).

MS, m/e (rel. intensity, %): 367 (3) [M+H—CN]⁺, 393 (100) [M+H]⁺, 410(10) [M+NH₄]⁺.

Example 12(E,E)-2-(Benzalaminocarbonyl)-3-(3,4-bis(t-butyldimethylsilyloxystyryl))acrylonitrile(CR18)

The compound was prepared as described in Example 3 by adding3,4-bis(t-butyldimethylsilyloxy)cinnamaldehyde (Example 11, 0.100 g,0.26 mmol) to N-(cyanoacetyl)benzylamide (Example 4, 0.044 g, 0.26 mmol.After refluxing for 2.5 h purification by silica gel chromatography(hexane-EtOAc, 15:1) provided a yellow solid (0.090 g, 64%). The productgave the following analytical data:

NMR (CD₃COCD₃, 8, ppm): 0.24 and 0.25 (2×s, 2×6H, Me₂Si+Me₂Si), 1.01 and1.02 (2×s, 2×9H, t-BuSi+t-BuSi), 4.55 (br.s., 2H, NHCH₂Ph), 7.00 (d, 1H,J=8.5 Hz, H⁴), 7.12 (dd, 1H, J 11.7 and 15.6 Hz, PhCCHCCN olefinic),7.24-7.43 (m, 8H, aromatic and olefinic protons), 7.93 (br.s., 1H, NH),8.02 (dd, 1H, J<0.5 and 11.7 Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 523 (30) [M+H—CN]⁺, 540 (24) [M+NH₄—CN]⁺,549 (89) [M+H]⁺, 566 (100) [M+NH₄]⁺.

Example 13(E,E)-2-(Benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile(CR4)—Method B

(E,E)-2-Benzylaminocarbonyl-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(Example 12, 0.028 g, 0.052 mmol) was treated with 60 μl of a 1M THFsolution of tetra-n-butylammonium fluoride in 2 ml of dry THF for 0.5 hat 20° C. After evaporation the compound was dissolved in 5 ml ofchloroform-methanol, 20:1, passed through silica gel and washed withchloroform-methanol, 20:1. The residue was purified by HPLCchromatography (MeCN—H₂O, 60:40, UV detection at 340 nm) leading to anorange solid (0.010 g, 62%). The analytical data were identical to thecompound prepared as described in Example 8.

Example 14 (E,E)-2-(3,4Dihydroxybenzylaminocarbonyl)-3-styrylacrylonitrile (CR19)

The compound was prepared as described in Example 3 by addingcinnamaldehyde (0.018 ml, 0.14 mmol) toN-(cyanoacetyl)3,4-dihydroxybenzylamide (Example 2, 0.03 g, 0.14 mmol).After refluxing for 2 h and recrystallization from ethanol, a yellowsolid was obtained (0.027 g, 59%). The product gave the followinganalytical data:

NMR (CD₃COCD₃, δ, ppm): 2.82 (br.s., 2H, (OH)₂), 4.39 (br.s., 2H,NHCH₂Ph), 6.70 (dd, 1H, J 1.9 and 8.2 Hz, H^(6′)), 6.76 (d, 1H, J=8.2Hz, H^(5′)), 6.87 (d, 1H, J=1.9 Hz, H^(2′)), 7.30 (dd, 1H, J 11.3 and15.7 Hz, PhCCHCCN olefinic), 7.47 and 7.73 (2×m, 6H, aromatic protonsand PhCH olefinic), 7.82 (br.s., 1H, NH), 8.04 (dd, 1H, J<0.5 and 11.3Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 321 (100) [M+H]⁺, 338 (65) [M+NH₄]⁺.

Example 15 (E,E)-2-(3,4Dihydroxybenzylaminocarbonyl)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(CR20)

The compound was prepared as described in Example 3 by adding3,4-bis(t-butyldimethylsilyloxy)cinnamaldehyde (Example 11, 0.015 g,0.038 mmol) to N-(cyanoacetyl)3,4-dihydroxybenzylamide (Example 2,0.0079 g, 0.038 mmol). After refluxing for 2 h and recrystallizationfrom ethanol a yellow solid was obtained (0.014 g, 64%). The productgave the following analytical data:

NMR (CD₃COCD₃, 8, ppm): 0.22 and 0.24 (2×s, 2×6H, Me₂Si+Me₂Si), 1.01 and1.03 (2×s, 2×9H, t-BuSi+t-BuSi), 2.72 (br.s., 2H, (OH)₂), 4.41 (br.s.,2H, NHCH₂Ph), 6.68-7.42 (m, 8H, aromatic and olefinic protons), 7.75(br.s., 1H, NH), 8.00 (dd, 1H, J<0.5 and 12.0 Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 555 (5) [M+H—CN]⁺, 572 (8) [M+NH₄—CN]⁺, 581(46) [M+H]⁺, 598 (100) [M+NH₄]⁺.

Example 16 (E,E)-2-(3,4Dihydroxybenzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile(CR21)

(E,E)-2-(3,4-Dihydroxybenzylaminocarbonyl)-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(Example 15, 0.026 g, 0.044 mmol) was treated with 60 μl of a 1M THFsolution of tetra-n-butylammonium fluoride in 1.5 ml of dry THF for 0.5h at 20° C. as described in Example 13. After purification, a yellowsolid was obtained (0.006 g, 43%). The product gave the followinganalytical data:

NMR (CD₃COCD₃, δ, ppm): 4.38 (s, 1H, NHCH₂Ph), 6.67-7.22 (m, 6H,Ph+Ph′), 7.05 (dd, 1H, J 11.8 and 15.5 Hz, PhC═CH olefinic), 7.34 (d,1H, J=15.5 Hz, PhCH olefinic), 7.70 (br.s, 1H, NH), 8.00 (d, 1H, J=11.8Hz, CH═CCN olefinic).

MS, m/e (rel. intensity, %): 186 (86) [(HO)₂C₆H₃CH═CHCH═CCN]⁺, 202 (28),242 (100) [M+H—C₆H₃(OH)₂]⁺, 353 (13) [M+H]⁺, 370 (6) [M+NH₄]⁺.

Example 17 (E,E)-2-(Benzylaminocarbonyl)-3-styrylacrylonitrile (CR1)

The compound was prepared as described in Example 3 by addingcinnamaldehyde (0.048 ml, 0.38 mmol) to N-(cyanoacetyl)benzylamide(Example 4, 0.066 g, 0.38 mmol). After refluxing for 1 h andrecrystallization from ethanol a white solid was obtained (0.074 g,68%). The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 4.55 (s, 1H, NHCH₂Ph), 7.24-7.51 (m, 11H,Ph+Ph′+PhCCHCCN olefinic), 7.72 (br.d, 1H, J=6.5 Hz, CHCN olefinic),7.98 (br.s., 1H, NH), 8.05 (d, 1H, J=11.7 Hz, PhCH olefinic).

MS, m/e (rel. intensity, %): 289 (100) [M+H]⁺, 306 (92) [M+NH₄]⁺.

Example 18(E,E)-2-(Benzylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR3)

The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.10 g, 0.48 mmol) toN-(cyanoacetyl)benzylamide (Example 4, 0.084 g, 0.48 mmol). Afterrefluxing for 3 h and recrystallization from ethanol, a yellow solid wasobtained (0.10 g, 57%). The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 3.90 (s, 6H, (OMe)₂), 4.55 (m, 2H, NHCH₂Ph),7.08 (br.s, 2H, H²⁺⁶), 7.17 (dd, 1H, J 11.5 and 15.2 Hz, PhCCHCCNolefinic), 7.22-7.41 (m, 6H, Ph′+PhCH olefinic), 7.90 (br.s., 1H, NH),8.01 (dd, 1H, J 0.55 and 11.7 Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 275 (14) [M+H—CN-MeOH-MeOH]⁺, 307 (9)[M+H—CN-MeOH]⁺, 339 (4) [M+H—CN]⁺, 365 (100) [M+H]⁺, 382 (16) [M+NH₄]⁺.

Example 19 N-(Cyanoacetyl)phenylpropylamide (A₅)

The compound was prepared as described in Example 1 by adding methylcyanoacetate (0.98 ml, 11.1 mmol) to phenylpropylamine (1.58 ml, 11.1mmol). The compound was distilled in vacuo directly from the reactionmixture (Kugelrohr apparatus (Aldrich), 0.1 mm Hg, T. oven 195-200° C.)to give an off-white solid (2.18 g, 97%). The product gave the followinganalytical data:

NMR (CD₃COCD₃, 5, ppm): 1.88 (q, 2H, J=7.3 Hz, PhCCH₂), 2.66 (t, 2H,J=7.3 Hz, PhCH₂), 3.28 (s, 2H, CNCH₂), 3.33 (dt, 2H, J 7.3 and 6.6 Hz,PhCCCH₂), δ 6.02 (br.s., 1H, NH), 7.15-7.30 (m, 5H, Ph).

MS, m/e (rel. intensity, %): 203 (88) [M+H]⁺, 220 (100) [M+NH₄]⁺.

Example 20 N-(Cyanoacetyl)phenylethylamide (A₄)

The compound was prepared as described in Example 1 by adding methylcyanoacetate (1.1 ml, 12.4 mmol) to phenylethylamine (1.55 ml, 12.4mmol). The compound was distilled in vacuo directly from the reactionmixture (Kugelrohr apparatus (Aldrich), 0.1 mm Hg, T. oven 190-195° C.)to give an off-white solid (2.14 g, 91%). The product gave the followinganalytical data:

NMR (CD₃COCD₃, δ, ppm): 2.80 (t, 2H, J=7.6 Hz, PhCH₂), 3.46 (br.t, 2H,J=7.6 Hz, PhCCH₂), 3.54 (s, 2H, CNCH₂), 7.20-7.31 (m, 5H, Ph), 7.51(br.s., 1H, NH).

MS, m/e (rel. intensity, %): 189 (100) [M+H]⁺, 206 (99) [M+NH₄]⁺.

Example 21(E,E)-2-(Phenylethylaminocarbonyl)-3-(3,4-dimethoxystyryl)acrylonitrile(CR5)

The compound was prepared as described in Example 3 by adding3,4-dimethoxycinnamaldehyde (Example 6, 0.1 g, 0.52 mmol) toN-(cyanoacetyl)phenylethylamide (Example 20, 0.1 g, 0.52 mmol). Afterrefluxing for 1 h and recrystallization from ethanol a yellow solid wasobtained (0.12 g, 63%). The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 2.91 (t, 2H, J=7.5 Hz, Ph′CH), 3.59 (br.t, 2H,J=7.5 Hz, Ph′CCH), 3.88, 3.89 (2×s, 2×3H, OCH₃+OCH₃), 7.04 (d, 1H, J=8.6Hz, H5), 7.16 (dd, 1H, J 11.8 and 15.0 Hz, PhC═CH olefinic), 7.20-7.42(m, 9H, aromatic+olefinic), 7.97 (d, 1H, J=11.8 Hz, CH═CCN olefinic).

MS, m/e (rel. intensity, %): 363 (100) [M+H]⁺, 380 (34) [M+NH₄]⁺.

Example 22(E,E)-2-(Phenylethylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR8)

The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.1 g, 0.48 mmol) toN-(cyanoacetyl)phenylethylamide (Example 20, 0.091 g, 0.48 mmol). Theresidue was purified by silica gel chromatography (CHCl₃-hexane, 1:1) togive a yellow solid (0.15 g, 83% yield). The product gave the followinganalytical data:

NMR (CD₃COCD₃, δ, ppm): 2.95 (t, 2H, J=7.6 Hz, CH₂Ph′), 3.62 (m, 2H,CH₂CPh′), 3.94 (s, 6H, (OMe)₂), 7.11 (s, 2H, H²⁺⁶), 7.19 (dd, 1H, J 11.7and 15.3 Hz, PhCCHCCN olefinic), 7.23-7.36 (m, 5H, Ph′), 7.41 (d, 1H,J=15.3 Hz, PhCH olefinic), 7.45 (br.s., 1H, NH), 7.99 (d, 1H, J=11.7 Hz,CHCN olefinic).

MS, m/e (rel. intensity, %): 379 (100) [M+H]⁺, 396 (7) [M+NH₄]⁺.

Example 23(E,E)-2-(Phenylpropylaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR9)

The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.10 g, 0.48 mmol) toN-(cyanoacetyl)phenylpropylamide (Example 19, 0.097 g, 0.48 mmol). Afterrefluxing for 3 h the residue was purified by silica gel chromatography(CHCl₃-hexane, 1:1) to give a brown solid (0.17 g, 90% yield). Theproduct gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 2.09 (q, 2H, J=7.5 Hz, NHCCH₂CPh′), 2.85 (t, 2H,J=7.5 Hz, CH₂Ph′), 3.57 (m, 2H, CH₂CPh′), 4.06 (s, 6H, (OMe)₂), 7.24 (s,2H, H²⁺⁶), 7.32 (dd, 1H, J 11.7 and 15.3 Hz, PhCCHCCN olefinic),7.33-7.46 (m, 5H, Ph′), 7.53 (d, 1H, J=15.3 Hz, PhCH olefinic), 7.58(br.s., 1H, NH), 8.11 (d, 1H, J=11.7 Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 331 (40), 348 (30), 359 (34), 376 (32), 393(100) [M+H]⁺, 410 (24) [M+NH₄]⁺.

Example 24(E,E)-2-Thioacetaminocarbonyl-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR12)

The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.15 g, 0.72 mmol) to2-cyanothioacetamide (0.073 g, 0.72 mmol). After refluxing for 1 h theresidue was purified on a TLC-plate in hexane-ethyl acetate, 1:1 to givea red solid (0.10 g, 52%). The product gave the following analyticaldata:

NMR (CD₃COCD₃, δ, ppm): 2.85 (br.s., OH+NH₂), 3.91 (s, 6H, (OMe)₂), 7.11(s, 2H, H²⁺⁶), 7.20 (dd, 1H, J 11.6 and 15.1 Hz, PhCCHCCN olefinic),7.46 (d, 1H, J=15.1 Hz, PhCH olefinic), 8.22 (dd, 1H, J 0.73 and 11.6Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 289 (100), 291 (60) [M+H]⁺, 312 (8)[M+Na]⁺.

Example 25(E,E)-2-Acetaminocarbonyl-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR13)

The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.1 g, 0.48 mmol) to2-cyanoacetamide (0.04 g, 0.48 mmol). After refluxing for 3 h andrecrystallization from ethanol an orange solid was obtained (0.083 g,63%). The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 2.82-2.88 (br.s., OH+NH₂), 3.90 (s, 6H, (OMe)₂),7.08 (s, 2H, H²⁺⁶), 7.16 (dd, 1H, J 11.6 and 15.1 Hz, PhCCHCCNolefinic), 7.38 (d, 1H, J=15.1 Hz, PhCH olefinic), 7.96 (dd, 1H, J 0.73and 11.6 Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 275 (100) [M+H]⁺, 292 (28) [M+NH₄]⁺.

Example 26 (E,E)-2-Carboxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile (CR14)

The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.15 g, 0.72 mmol) to cyanoaceticacid (0.061 g, 0.72 mmol). After refluxing for 1 h and recrystallizationfrom ethanol a yellow solid was obtained (0.15 g, 75%). The product gavethe following analytical data:

NMR (CD₃COCD₃, δ, ppm): 3.00 (br.s., OH), 3.91 (s, 6H, (OMe)₂), 7.12 (s,2H, H²⁺⁶), 7.21 (dd, 1H, J 11.6 and 15.1 Hz, PhCCHCCN olefinic), 7.50(d, 1H, J=15.1 Hz, PhCH olefinic), 8.04 (dd, 1H, J 0.73 and 11.6 Hz,CHCN olefinic).

MS, m/e (rel. intensity, %): 276 (66) [M+H]⁺, 293 (100) [M+NH₄]⁺.

Example 27(E,E)-2-Carbomethoxy-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR15)

The compound was prepared as described in Example 3 by adding3,5-dimethoxy-4-hydroxycinnamaldehyde (0.15 g, 0.72 mmol) to methylcyanoacetate (0.064 ml, 0.72 mmol). After refluxing for 1 h andrecrystallization from ethanol an orange solid was obtained (0.2 g,90%). The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 2.84 (br.s., OH), 3.84 (s, 3H, COOMe), 3.91 (s,6H, (OMe)₂), 7.12 (s, 2H, H²⁺⁶), 7.21 (dd, 1H, J 11.6 and 15.1 Hz,PhCCHCCN olefinic), 7.53 (d, 1H, J=15.1 Hz, PhCH olefinic), 8.05 (dd,1H, J 0.73 and 11.6 Hz, CHCN olefinic).

MS, m/e (rel. intensity, %): 290 (100) [M+H]⁺, 307 (99) [M+NH₄]⁺.

Example 28(E,E)-2-Acetaminocarbonyl-3-[3,4-bis(t-butyldimethylsilyloxystyryl)]acrylonitrile(CR16)

The compound was prepared as described in Example 3 by adding3,4-bis(t-butyldimethylsilyloxy)cinnamaldehyde (Example 11, 0.15 g, 0.38mmol) to 2-cyanoacetamide (0.032 g, 0.38 mmol). After refluxing for 0.5h, purification by silica gel chromatography (hexane-EtOAc, 5:1)provided a crystallizing oil (0.10 g, 57%).

NMR (CD₃COCD₃, δ, ppm): 0.22 and 0.24 (2×s, 2×6H, Me₂Si+Me₂Si), 1.01 and1.03 (2×s, 2×9H, t-BuSi+t-BuSi), 7.01, 7.23-7.29 (m, 3H, aromatic), 7.11(dd, 1H, J 11.9 and 15.3 Hz, PhC═CH olefinic), 7.40 (d, 1H, J=15.3 Hz,PhCH olefinic), 7.98 (d, 1H, J=11.9 Hz, CH═CCN olefinic).

MS, m/e (rel. intensity, %): 459 (100) [M+H]⁺, 476 (89) [M+NH₄]⁺.

Example 29(E,E)-2-Acetaminocarbonyl-3-(3,4-dihydroxystyryl)acrylonitrile (CR17)

(E,E)-2-Acetaminocarbonyl-3-(3,4-bis(t-butyldimethylsilyloxystyryl))acrylonitrile(Example 28, 0.1 g, 0.22 mmol) was treated with an excess of a 1M THFsolution of tetra-n-butylammonium fluoride in benzene for 0.5 h at 20°C. as described in Example 13. After purification, a yellow solid wasobtained (0.04 g, 85%). The product gave the following analytical data:

MS, m/e (rel. intensity, %): 231 (83) [M+H]⁺, 248 (100) [M+NH₄]⁺.

Example 30 N-(Cyanoacetyl)β-ethanolamide (A₁₁)

To β-ethanolamine (1.37 ml, 22.6 mmol), methyl cyanoacetate was added(2.0 ml, 22.6 mmol). The reaction was heated for 30 h at 100° C. Coolinggave a brown solid which was recrystallized from ethanol to give 2.10 gof the product (71%). The product gave the following analytical data:

MS, m/e (rel. intensity, %):129 (30) [M+H]⁺, 146 (100) [M+NH₄]⁺.

Example 31(E,E)-2-(β-Ethanolaminocarbonyl)-3-(3,5-dimethoxy-4-hydroxystyryl)acrylonitrile(CR24)

To 3,5-dimethoxy-4-hydroxycinnamaldehyde (0.018 g, 0.086 mmol),N-(cyanoacetyl)β-ethanolamide (Example 30, 0.010 g, 0.086 mmol) wasadded. After refluxing for 2 h and purification on silica gel,CHCl₃-MeOH, 5:1, a yellow solid was obtained. (0.024 g, 87%). Theproduct gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 3.47, 3.67 (2×m, 4H, NHCH₂+CH₂OH), 3.90 (s, 6H,OCH₃+OCH₃), 7.07 (br.s, 2H, H²⁺⁶), 7.16 (dd, 1H, J 11.7 and 15.2 Hz,PhC═CH olefinic), 7.31 (br.s, 1H, NH), 7.38 (d, 1H, J=15.2 Hz, PhCHolefinic), 7.97 (d, 1H, J=11.7 Hz, CH═CCN olefinic).

MS, m/e (rel. intensity, %): 319 (70) [M+H]⁺, 341 (100) [M+Na]⁺.

Example 32 (E,E)-2-(Benzylaminocarbonyl)-3-(4-nitrostyryl)acrylonitrile(CR27)

The compound was prepared as described in Example 3 by adding4-nitrocinnamaldehyde (0.022 g, 0.12 mmol) to N-(cyanoacetyl)benzylamide(Example 4, 0.022 g, 0.12 mmol). After refluxing for 1 h, the productwas purified by silica gel chromatography (CHCl₃-MeOH, 5:1) to give ayellow solid (0.033 g, 81%). The product gave the following analyticaldata:

NMR (CD₃COCD₃, δ, ppm): 4.56 (br.s, 2H, NHCH₂), 7.24-7.38 (m, 6H,Ph′+NH), 7.47 (dd, 1H, J 11.1 and 15.2 Hz, PhC═CH olefinic), 7.62 (d,1H, J=15.2 Hz, PhCH olefinic), 8.02, 8.32 (2×br.d, 4H, J 8.8 and 8.3 Hz,Ph), 8.08 (d, 1H, J=11.1 Hz, CH═CCN olefinic).

MS, m/e (rel. intensity, %): 334 (100) [M+H]⁺, 351 (16) [M+NH₄]⁺, 356(28) [M+Na]⁺.

Example 33(E,E)-2-(3,4-Dihydroxybenzylaminocarbonyl)-3-(4-nitrostyryl)acrylonitrile(CR28)

The compound was prepared as described in Example 3 by adding4-nitrocinnamaldehyde (0.009 g, 0.05 mmol) toN-(cyanoacetyl)3,4-dihydroxybenzylamide (Example 2, 0.010 g, 0.05 mmol).After refluxing for 2 h and recrystallization from ethanol a yellowsolid was obtained (0.007 g, 39%). The product gave the followinganalytical data:

NMR (CD₃COCD₃, 8, ppm): 2.81, 2.83 (2×br.s, 2H, OH+OH), 4.39 (br.s, 2H,NHCH₂), 6.69 (br.d, 1H, J<0.5 and 7.6 Hz, H⁶), 6.76 (d, 1H, J=7.6 Hz,H^(5′)), 6.86 (br.d, 1H, J<0.5 Hz, H^(2′)), 7.47 (dd, 1H, J 11.7 and15.2 Hz, PhC═CH olefinic), 7.61 (d, 1H, J=15.2 Hz, PhC olefinic), 7.91(br.s, 1H, NH), 8.02, 8.31 (2×br.d, 4H, J 8.2 and 8.8 Hz, Ph), 8.06 (d,1H, J=11.7 Hz, CH═CCN olefinic).

MS, m/e (rel. intensity, %): 331 (21) [M-OH—OH]⁺, 348 (47) [M-OH]⁺, 366(100) [M+H]⁺, 383 (97) [M+NH₄]⁺.

Example 34(E,E)-2-(1-Amino-2,2-dicyanoethenyl)-3-(4-nitrostyryl)acrylonitrile(CR29)

The compound was prepared as described in Example 3 by adding4-nitrocinnamaldehyde (0.051 g, 0.29 mmol) to2-amino-1-propene-1,1,3-tricarbonitrile (0.038 g, 0.29 mmol). Afterrefluxing for 4 h and recrystallization from ethanol a yellow solid wasobtained (0.08 g, 51%). The product gave the following analytical data:

NMR (CD₃COCD₃, δ, ppm): 7.54 (dd, 1H, J 11.1 and 15.8 Hz, PhC═CHolefinic), 7.67 (d, 1H, J=15.8 Hz, PhCH olefinic), 7.99 (d, 1H, J=11.1Hz, CH═CCN olefinic), 8.08, 8.32 (2×br.d, 4H, J 8.8 and 8.8 Hz, Ph).

MS, m/e (rel. intensity, %): 309 (100) [M+NH₄]⁺, 314 (67) [M+Na]⁺.

Example 35 Effect of CR4 Upon Normal Bone Marrow Differentiation inCulture

The CFU-GEMM assay was performed according to Fauser and Messner (1978,Blood, 52(6) 143-8) and Messner and Fausser (1980, Blut, 41(5) 327-33)with some variations. In brief, heparinized bone marrow cells werelayered over Percoll (1.077 gm/ml) (Pharmacia Fine Chemical, PiscatawayN.J.) and centrifuged at 400 g at 4° C. for 10 minutes to removeneutrophils and RBCs. The fractionated BM cells at 2×10⁵ cells/ml werecultured in IMDM (OCI, Toronto) containing 0.9% (vol/vol)methylcellulose supplementd with 30% FCS (Cansera Rexdale, ON.) ornormal human plasma, a cocktail of cytokines containing G-CSF (10 ng/ml,Amgen), IL-3 (40 U/ml, Immunex), MGF (50 ng/ml, Immunex), Erythropoietin(2 u/ml, Epprex) or TPO (10 ng/ml, Amgen), 5×10⁻⁵M β-2-mercaptoethanoland the specified concentration of CR4. The culture mixture was platedin 1 ml volumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂in a humidified atmosphere. All cultures were evaluated at 14 days forthe number of BFU-E colonies (defined as aggregates of more than 500hemoglobinized cells or, 3 or more erythroid subcolonies), CFU-GMcolonies (defined as granulocyte or monocyte-macrophage cells or both),CFU-Meg colonies (comprising 4 or more megakaryocytes) and CFU-GEMMcolonies (a mixed population comprising of all elements).

The results shown in FIG. 1 demonstrate that CR4 displayed negligibletoxicity upon normal bone marrow at doses up to 5 μM. At 10 μM CR4 beganto cause some inhibition of BFU-E colony formation, but at the same timesignificantly stimulated CFU-GM colony numbers.

Example 36 Killing of Philadelphia Positive Acute Lymphoblastic Leukemiaby Low-Dose CR4 in Culture

Ph+ ALL cells were plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing alphaMEM (Gibco) plus 10-20% FCS (Cansera Rexdale, ON.) in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland) with the indicated concentrationsof CR4. Cultures were set at 37° C., 5% CO₂ in a humidified atmosphere.Colonies consisting of more than 20 cells were counted at 12 days orearlier using an inverted microscope.

The results shown in FIG. 2 demonstrate that CR4 effected a significantinhibition of Ph+ ALL cell proliferation and survival at low nanomolardoses (35-100). CR4 has no effect upon normal cells at equivalentconcentrations.

Example 37 Killing of Philadelphia Positive Z119 Acute LymphoblasticLeukemia Cells by Low-Dose CR4 in Culture

Z119 cells were plated in 1 ml volumes at a density of 1×10⁴ cells/ml,in the absence of exogenous growth factors, into 35 mm petri dishes(Nunc, Gibco) containing IMDM (OCI, Toronto) plus 20% FCS (CanseraRexdale, ON.) in 0.9% (vol/vol) methylcellulose (Fluka, Switzerland)with the indicated concentration of CR4. Cultures were set at 37° C., 5%CO₂ in a humidified atmosphere. Colonies consisting of more than 20cells were counted at 7 days or earlier using an inverted microscope.

The results shown in FIG. 3 demonstrate that CR4 effected a significantinhibition of Z119 ALL cell proliferation and survival at low nanomolardoses. CR4 has no effect upon normal cells at equivalent concentrations.

Example 38 Killing of AML-3 Acute Myeloid Leukemia Cells by Low-Dose CR4in Culture

OCI-AML-3 cells were plated in 35 mm petri dishes (Nunc, Gibco) in 1 mlvolumes at a density of 3.3×10³ cells/ml, in the absence of exogenousgrowth factors, containing alpha MEM plus 20% FCS (Cansera, RexdaleOnt.), and 0.9% (vol/vol) methylcellulose (Fluka, Switzerland) and theindicated concentrations of CR4. Cell cultures were incubated in ahumidified atmosphere at 37° C. with 5% CO₂. Colonies containing morethan 20 cells were scored, using an inverted microscope, at 5-6 days.

The results shown in FIG. 4 demonstrate that CR4 effected a completeinhibition of AML-3 cell proliferation and survival at nanomolarconcentrations (300-600 nM). CR4 has no effect upon normal cell survivalat equivalent concentrations.

Example 39 Killing of Ly-MN Lymphoma Cells by Low-Dose CR4 in Culture

Ly-MN cells were plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing IMDM(OCI, Toronto) plus 20% human cord blood plasma in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland) and the indicated concentrations ofCR4. Cultures were set at 37° C., 5% CO₂ in a humidified atmosphere.Colonies consisting of more than 20 cells were counted at 5 days orearlier using an inverted microscope.

The results shown in FIG. 5 demonstrate that CR4 significantly inhibitedcell proliferation and survival at nanomolar doses, and effected ainhibition by 2.5 μM. CR4 has no effect upon normal cells at equivalentconcentrations.

Example 40 Killing of Primary Juvenile Myelo-Monocytic Leukemia Cells byCR4 in Culture

Heparinized bone marrow cells from a JMML patient were layered overPercoll (1.077 gm/ml) (Pharmacia Fine Chemical, Piscataway N.J.) andcentrifuged at 400 g at 4° C. for 10 minutes to remove neutrophils andRBCs. Cells were further fractionated and purified on Miltenyi MScolumns (Miltenyi Biotec GmbH, Germany) to acquire an early progenitorpopulation of CD34⁺ cells. The fractionated BM CD34⁺ cells at a densityof 1×10⁴ cells/ml were cultured in IMDM (OCI, Toronto) containing 0.9%(vol/vol) methylcellulose supplemented with 30% FCS (Cansera Rexdale,ON.) with the indicated concentration of CR4. The culture mixture wasplated in 1 ml volumes into 35 mm petri dishes and incubated at 37° C.,5% CO₂ in a humidified atmosphere. Colonies consisting of more than 20cells were counted at 12 days or earlier using an inverted microscope.

The results shown in FIG. 6 demonstrate that CR4 displayed moderatekilling ability with primary JMML cells, with 80-90 percent inhibitionachieved by 5 μM concentrations.

Example 41 Killing of OCI-LY2 Lymphoma Cells by Low-Dose CR4 in Culture

OCI-LY2 cells were plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing IMDM(OCI, Toronto) plus 20% human cord blood plasma in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland) and the indicated doses of CR4.Cultures were set at 37° C., 5% CO₂ in a humidified atmosphere. Coloniesconsisting of more than 20 cells were counted at 5 days or earlier usingan inverted microscope.

The results shown in FIG. 7 demonstrate that CR4 significantly inhibitedcell proliferation and survival at high nanomolar to low micromolardoses (90% at 2.5 μM). CR4 has no effect upon normal cells at equivalentconcentrations.

Example 42 Killing of Philadelphia Positive ALL Cells by CR17 and CR21in Culture

ALL cells were plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing alphaMEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland) and the indicated concentrations ofcompound. Cultures were set at 37° C., 5% CO₂ in a humidifiedatmosphere. Colonies consisting of more than 20 cells were counted at 12days or earlier using an inverted microscope.

The results shown in FIG. 8 demonstrate that CR17 displayed significantinhibition of cell growth at 1-2.5 μM concentrations. CR21 inhibitedcell growth at 5 μM.

Example 43 Killing of Philadelphia Positive ALL Cells by CR17 and CR21in Culture

ALL cells were plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing alphaMEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland) and the indicated concentrations ofcompound. Cultures were set at 37° C., 5% CO₂ in a humidifiedatmosphere. Colonies consisting of more than 20 cells were counted at 12days or earlier using an inverted microscope.

The results shown in FIG. 9 demonstrate that CR17 and CR21 bothdisplayed significant inhibition of cell growth at 1-2.5 μMconcentrations.

Example 44 Killing of Philadelphia Positive ALL Cells by CR24 in Culture

ALL cells were plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing alphaMEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland) and the indicated concentrations ofCR24. Cultures were set at 37° C., 5% CO₂ in a humidified atmosphere.Colonies consisting of more than 20 cells were counted at 9 days orearlier using an inverted microscope.

The results shown in FIG. 10 demonstrate that CR24 was effective againstPh+ ALL cells at concentrations as low as 0.5 μM, demonstrating avirtually complete inhibition of cell growth between 2.5 and 5 μM.

Example 45 Killing of Philadelphia Positive ALL Cells by CR19 in Culture

ALL cells were plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing alphaMEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland) and the indicated concentrations ofCR19. Cultures were set at 37° C., 5% CO₂ in a humidified atmosphere.Colonies consisting of more than 20 cells were counted at 9 days orearlier using an inverted microscope.

The results shown in FIG. 11 demonstrate that CR19 was highly effectiveagainst Ph+ ALL cells at nanomolar concentrations between 250 and 500nM.

Example 46 Effect of CR19 on Normal Bone Marrow Differentiation inCulture

The CFU-GEMM assay was performed according to Fauser and Messner (1978,Blood, 52(6) 1243-8) and Messner and Fausser (1980, Blut, 41(5) 327-33)with some variations. In brief, heparinized bone marrow cells werelayered over Percoll (1.077 gm/ml) (Pharmacia Fine Chemical, PiscatawayN.J.) and centrifuged at 400 g at 4° C. for 10 minutes to removeneutrophils and RBCs. The fractionated BM cells at 2×10⁵ cells/ml werecultured in IMDM (OCI, Toronto) containing 0.9% (vol/vol)methylcellulose supplementd with 30% FCS (Cansera Rexdale, ON.) ornormal human plasma, a cocktail of cytokines containing G-CSF (10 ng/ml,Amgen), IL-3 (40 U/ml, Immunex), MGF (50 ng/ml, Immunex), Erythropoietin(2 u/ml, Epprex) or TPO (10 ng/ml, Amgen), 5×10⁻⁵M β-2-mercaptoethanoland the specified concentrations of CR19. The culture mixture was platedin 1 ml volumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂in a humidified atmosphere. All cultures were evaluated at 14 days forthe number of BFU-E colonies (defined as aggregates of more than 500hemaglobinized cells or, 3 or more erythroid subcolonies) and CFU-Ccolonies (defined as granulocyte or monocyte-macrophage cells or both).

The results shown in FIG. 12 demonstrate that CR19 displayed significantinhibition of the development of BFU-E colonies at 2.5 μM, although atthis concentration it also boosted CFU-C colony formation. At 5 μM thestimulatory effect disappeared and BFU-E colonies were virtually absent.

Example 47 Effect of CR24, CR17 and CR21 on Normal Bone MarrowDifferentiation

The CFU-GEMM assay was performed according to Fauser and Messner (1978,Blood, 52(6) 1243-8) and Messner and Fausser (1980, Blut, 41(5) 327-33)with some variations (British Journal of Haematology, 1992, 80, p40-48). In brief, heparinized bone marrow cells were layered overPercoll (1.077 gm/ml) (Pharmacia Fine Chemical, Piscataway N.J.) andcentrifuged at 400 g at 4° C. for 10 minutes to remove neutrophils andRBCs. The fractionated BM cells at 2×10⁵ cells/ml were cultured in IMDM(OCI, Toronto) containing 0.9% (vol/vol) methylcellulose supplementdwith 30% FCS (Cansera Rexdale, ON.) or normal human plasma, a cocktailof cytokines containing G-CSF (10 ng/ml, Amgen), IL-3 (40 U/ml,Immunex), MGF (50 ng/ml, Immunex), Erythropoietin (2 u/ml, Epprex) orTPO (10 ng/ml, Amgen), 5×10⁻⁵M β-2-mercaptoethanol and the specifiedconcentration of test compound. The culture mixture was plated in 1 mlvolumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂, in ahumidified atmosphere. All cultures were evaluated at 14 days for thenumber of BFU-E colonies (defined as aggregates of more than 500hemoglobinized cells or, 3 or more erythroid subcolonies) and CFU-Ccolonies (defined as granulocyte or monocyte-macrophage cells or both).

The results shown in FIG. 13 demonstrate that CR24 displayed minimalinhibition of bone marrow colony formation at either 10 or 20 μMconcentrations, whereas both CR17 and CR21 caused inhibition of BFU-Ecolony formation at the higher 20 μM dose.

Example 48 In Vitro Purging of Normal Bone Marrow with CR4

Heparinized bone marrow cells were layered over Percoll (1.077 gm/ml)(Pharmacia Fine Chemical, Piscataway N.J.) and centrifuged at 400 g at4° C. for 10 minutes to remove neutrophils and RBCs.

For the purging process, the cells were resuspended at 1×10⁶/ml incomplete medium with or without 50 μM CR4. The cells were incubated withthe CR4 for two and a half hours at 37° C., 5% CO₂. At the end of thisperiod the cells were thoroughly washed in medium to remove CR4 and thencultured at 2×10⁵ cells/ml in IMDM (OCI, Toronto) containing 0.9%(vol/vol) methylcellulose supplemented with 30% FCS (Cansera Rexdale,ON.) or normal human plasma, a cocktail of cytokines containing G-CSF(10 ng/ml, Amgen), IL-3 (40 U/ml, Immunex), MGF (50 ng/ml, Immunex),Erythropoietin (2 u/ml, Epprex) or TPO (10 ng/ml, Amgen) and 5×10⁻⁵Mβ-2-mercaptoethanol. The culture mixture was plated in 1 ml volumes into35 mm petri dishes and incubated at 37° C., 5% CO₂ in a humidifiedatmosphere. All cultures were evaluated at 14 days for the number ofBFU-E colonies (defined as aggregates of more than 500 hemaglobinizedcells or, 3 or more erythroid subcolonies), CFU-C colonies (defined asgranulocyte or monocyte-macrophage cells or both) and CFU-GEMM colonies(a mixed population comprising of all elements).

The results shown in FIG. 14 demonstrate that two and a half hoursexposure to 50 μM CR4 did not result in significant inhibition of colonyformation. While a slight drop in BFU-E colonies occurred, CFU-C colonynumbers actually increased significantly.

Example 49 In Vitro Purging of Z119 Acute Lymphoblastic Leukemia withCR4

For the purging assay, the cells were resuspended in complete mediumwith or without CR4 as indicated and incubated at 37° C., 5% CO₂ for 0-5hours. Cells were then washed thoroughly with medium to remove the CR4,resuspended and plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing alphaMEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland). Cultures were set at 37° C., 5%CO₂ in a humidified atmosphere. Colonies consisting of more than 20cells were counted at 9 days or earlier using an inverted microscope.

The results shown in FIG. 15 demonstrate that CR4 demonstrated rapidkilling of Z119 cells at the concentrations examined. 50 μM CR4displayed a complete inhibition after only 2.5 hours exposure, while 85%killing could be achieved with 25 μM CR4 over the same time period. Alonger five hour exposure of the cells to 25 μM CR4 resulted in acomplete ablation of subsequent cell growth.

Example 50 In Vitro Purging of OCI-Ly2 Lymphoma Cells with CR4

For the purging assay, the cells were resuspended in complete mediumwith or without CR4 as indicated and incubated at 37° C., 5% CO₂ for 0-5hours. Cells were then washed thoroughly with medium to remove CR4,resuspended and plated in 1 ml volumes at 5×10³ cells/ml, in the absenceof exogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing IMDM (OCI, Toronto) plus 20% human cord blood plasma in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland). Cultures were set at 37°C., 5% CO₂ in a humidified atmosphere. Colonies consisting of more than20 cells were counted at 5 days or earlier using an inverted microscope.

The results shown in FIG. 16 demonstrate that CR4 demonstratedsignificant killing (90%) of OCI-Ly2 cells at 25-50 μM after only 5hours exposure. The lower 12.5 μM dose tested also achieved significantkilling in the same time period.

Example 51 In Vitro Purging of OCI-AML-3 Acute Meyloid Leukemia Cellswith CR4

For the purging assay, the cells were resuspended in complete mediumwith or without CR4 as indicated and incubated at 37° C., 5% CO₂ for 0-5hours. Cells were then washed thoroughly with medium to remove CR4,resuspended and plated in 1 ml volumes at 5×10³ cells/ml, in the absenceof exogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 10% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland). Cultures were set at 37°C., 5% CO₂ in a humidified atmosphere. Colonies consisting of more than20 cells were counted at 5 days or earlier using an inverted microscope.

The results shown in FIG. 17 demonstrate that CR4 demonstratedsignificant killing of OCI-AML-3 cells at 50 μM after only 2.5-5 hoursexposure. The lower 25 μM dose tested also achieved significant killingin the same time period.

Example 52 In Vitro Purging of Ramos B Cell Burkitt's Lymphoma Cellswith CR4

For the purging assay, the cells were resuspended in complete mediumwith or without CR4 as indicated and incubated at 37° C., 5% CO₂ for 0-5hours. Cells were then washed thoroughly with medium to remove the CR4,resuspended and plated in 1 ml volumes, in the absence of exogenousgrowth factors, into 35 mm petri dishes (Nunc, Gibco) containing RPMI1640 (Gibco) plus 10% FCS (Cansera Rexdale, ON.) in 0.9% (vol/vol)methylcellulose (Fluka, Switzerland). Cultures were set at 37° C., 5%CO₂ in a humidified atmosphere. Colonies consisting of more than 20cells were counted at 12 days or earlier using an inverted microscope.

The results shown in FIG. 18 demonstrate that CR4 demonstratedsignificant killing (70%) of Ramos cells at 50 μM after 5 hoursexposure.

Example 53 Killing of HuNS1 Multiple Myeloma by CR4

HuNS1 cells were plated in 35 mm petri dishes (Nunc, Gibco) in 1 mlvolumes at a density of 1×10⁴ cells/ml, in the absence of exogenousgrowth factors, containing alpha MEM plus 20% FCS (Cansera, RexdaleOnt.), and 0.9% (vol/vol) methylcellulose (Fluka, Switzerland) and theindicated concentrations of CR4. Cell cultures were incubated in ahumidified atmosphere at 37° C. with 5% CO₂. Colonies containing morethan 20 cells were scored, using an inverted microscope, at 5-6 days.

The results shown in FIG. 19 demonstrate that CR4 significantlyinhibited cell proliferation and survival at high nanomolar to lowmicromolar doses (>90% at 2.5 μM). CR4 has no effect upon normal cellsat equivalent concentrations.

Example 54 In Vivo Treatment of Philadelphia Positive AcuteLymphoblastic Leukemia in NOD-SCID Mice

NOD-SCID mice were irradiated (350 rads) and injected with 5×10⁶Philadelphia positive Z119 Acute lymphoblastic leukemia cells. After 24hours Alzet micro-osmotic pumps (Alza Corp. Paolo Alto, Calif.) wereimplanted subcuntaneously, containing either 20 mM solution of CR4 in50% DMSO/medium or 50% DMSO/medium alone. Alzet 2001 pumps wereutilized, holding a total volume of 200 μl and releasing 1 μl per hourover 7-10 days. Pumps were replaced after 7 days. Each mouse received adaily dose of 0.154 mg of CR4.

After 14 (FIG. 20A) and 21 (FIG. 20B) days mice were sacrificed and bonemarrow extracted from the fore and hind limbs. Single cell suspensionswere prepared, red blood cells lysed and the samples stained withPE-labelled isotype, anti-human CD19 and anti-human HLA-DR antibodies todetect the presence of Z119 cells. These antibodies do not cross reactwith murine cells.

At d14, bone marrow cell cultures were also performed to assess thepresence of Z119 cells. 5×10⁴ BM cells were cultured in IMDM (OCI,Toronto) containing 0.9% (vol/vol) methylcellulose supplemented with 30%serum consisting of a 1:1 mixture of FCS (Cansera Rexdale, ON.) andnormal human plasma. No cytokines are added. Under these conditionsthere is no growth of murine cells. The culture mixture was plated in 1ml volumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂ in ahumidified atmosphere. All cultures were evaluated at 9 days for thenumber of ALL colonies.

The results shown in FIGS. 20A and 20B demonstrate that at both day 14and 21 sacrifices, a significant reduction in ALL infiltration of thebone marrow was observed in all the mice treated with CR4 relative tothe DMSO treated control mice.

In control animals, massive infiltration of the spleen, liver and kidneywas observed, as well as the presence of ALL cells in the peripheralblood. In addition to a 90% reduction in the infiltration of ALL cellsinto the bone marrow, treatment with CR4 reduced ALL infiltration of theorgans and blood to below detectable levels.

Thus, CR4 was highly effective against a variety of cancer cells,including acute lymphoblastic leukemia, Philadelphia positive ALL, acutemyeloid leukemia, myeloma and B-lineage lymphoma, at concentrationsranging from 50 nM to 5 μM. At the same time, minimal toxicity was seenwhen normal cells were incubated in the presence of CR4 untilconcentrations of 10-20 μM or greater were achieved. CR4 wasparticularly active against bcr-abl transformed Philadelphia positivecells, achieving >90% wipeout at concentrations as low as 40 nM. CR4 wasalso highly effective in high dose (25-50 μM) in vitro purging assaysagainst Philadelphia positive ALL, AML and lymphoma, causing >90%inhibition of growth with a 2.5 to 5 hour exposure time. Over identicaldoses and times normal bone marrow growth and differentiation wereunaffected. CR4 showed a combination of high level toxicity to cancercells with minimal non-specific cytotoxic damage.

CR4 was also highly effective in a whole animal model (Example 54). Thecompound demonstrated good retention characteristics, still beingdetectable in the blood 30 minutes after I.V. injection. In a murinemodel of human Ph+ ALL, CR4 caused a greater than 90 percent reductionin ALL infiltration of bone marrow within a two week period, reducingthe presence of infiltrating ALL cells in liver, kidney, spleen andperipheral blood below detection level. In contrast, control micetreated with vehicle alone demonstrated massive infiltration of allthese organs. No evidence of non-specific toxicity was observed.

Example 55 In Vitro Purging of Normal Bone Marrow with CR11

Heparinized bone marrow cells were layered over Percoll (1.077 gm/ml)(Pharmacia Fine Chemical, Piscataway N.J.) and centrifuged at 400 g at4° C. for 10 minutes to remove neutrophils and RBCs.

For the purging process, the cells were resuspended at 1×10⁶/ml incomplete medium with or without 50 μM CR11. The cells were incubatedwith the tryrphostin for seven hours at 37° C., 5% CO₂. At the end ofthis period the cells were thoroughly washed in medium to remove CR11and then cultured at 2×10⁵ cells/ml in IMDM (OCI, Toronto) containing0.9% (vol/vol) methylcellulose supplementd with 30% FCS (CanseraRexdale, ON.) or normal human plasma, a cocktail of cytokines containingG-CSF (10 ng/ml, Amgen), IL-3 (40 U/ml, Immunex), MGF (50 ng/ml,Immunex), Erythropoietin (2 u/ml, Epprex) or TPO (10 ng/ml, Amgen) and5×10⁻⁵M β-2-mercaptoethanol. The culture mixture was plated in 1 mlvolumes into 35 mm petri dishes and incubated at 37° C., 5% CO₂ in ahumidified atmosphere. All cultures were evaluated at 14 days for thenumber of BFU-E colonies (defined as aggregates of more than 500hemoglobinized cells or, 3 or more erythroid subcolonies), CFU-Ccolonies (defined as granulocyte or monocyte-macrophage cells or both)and CFU-GEMM colonies (a mixed population comprising of all elements).

The results shown in FIG. 21 demonstrate that seven hours exposure to 50μM CR11 did not result in any significant inhibition of colonyformation. BFU-E, CFU-GEMM and CFU-C colonies were all normal.

Example 56 In Vitro Purging of Philadelphia Positive Acute LymphoblasticLeukemia with CR11

For the purging assay, the cells were resuspended in complete mediumwith or without CR11 as indicated and incubated at 37° C., 5% CO₂ for0-7 hours. Cells were then washed thoroughly with medium to remove theCR11, resuspended and plated in 1 ml volumes, in the absence ofexogenous growth factors, into 35 mm petri dishes (Nunc, Gibco)containing alpha MEM (Gibco) plus 20% FCS (Cansera Rexdale, ON.) in 0.9%(vol/vol) methylcellulose (Fluka, Switzerland). Cultures were set at 37°C., 5% CO₂ in a humidified atmosphere. Colonies consisting of more than20 cells were counted at 9 days or earlier using an inverted microscope.

The results shown in FIG. 22 demonstrate that CR11 demonstrated completekilling of Ph+ ALL cells at 50 μM after 7 hours exposure.

Example 57 Philadelphia (Ph+) ALL Lines Z119 and Z181 (5×10⁶Cells/Point) were Lysed and Immunoprecipitated with Bcr-Abl Antibody

The precipitates were washed twice with lysis buffer and once withkinase assay buffer, and resuspended in same buffer containing varyingconcentrations of CR4. The precipitates were incubated with the drug for10 min at room temperature, followed by addition of 10 μCi ³³PγATP. Thereaction was stopped after 20 min by the addition of SDS-PAGE reducingsample buffer and separated on an 8-16% SDS-PAGE gel. The products weretransferred onto nitrocellulose membrane and visualized byautoradiography.

The results shown in FIG. 23 demonstrate that Bcr-Abl kinase activity iseffectively blocked at concentrations of 1 to 10 μM of the CR4 compoundin both Z119 and Z181 ALL cell lines.

Example 58 Philadelphia (Ph+) ALL Line Z119 (5×10⁶ Cells/Point) wasPreincubated for 5 Hours with Different Concentrations of CR4 andImmunoprecipitated with Jak2 Antibody

The cells were lysed in lysis buffer and immunoprecipitated with Jak2antibody. The precipitates were washed twice with lysis buffer and oncewith kinase assay buffer, followed by addition of 10 μCi ³³PγATP. Thereaction was stopped after 20 min by the addition of SDS-PAGE reducingsample buffer and separated on an 8-16% SDS-PAGE gel. The products weretransferred onto nitrocellulose membrane and visualized byautoradiography.

The results shown in FIG. 24 demonstrate that Jak2 kinase activity wasdramatically inhibited at a concentration of 6 μM and further blocked athigher concentrations.

Example 59(E,E)-[2-Cyano-5-(3,4-dihydroxyphenyl)-penta-2,4-dienoyl]carbamic acidethyl ester

The compound was prepared as described in Example 3 by adding3,4-dihydroxycinnamaldehyde (32 mg, 0.2 mmol) to N-cyanoacetylurethane(32 mg, 0.2 mmol). After refluxing for 1 h and recrystallization fromethanol-water an orange solid was obtained (54 mg, 90%). The productgave the following analytical data:

NMR (CD₃C(O)CD₃, 8, ppm): 1.28; 4.21 (t and q, J=7.1 Hz, COOEt), 6.92(d, 1H, J=8.2 Hz, H⁵), 7.08 (dd, 1H, J 11.5 and 15.2 Hz, PhC═CH), 7.12(dd, 1H, J 2.2 and 8.2 Hz, H⁶), 7.25 (d, 1H, J=2.2 Hz, H²), 7.44 (d, 1H,J=15.2 Hz, PhCH═C), 8.08 (d, 1H, J=11.5 Hz, CH═CCN).

MS, m/e (rel. intensity, %): 303 (100) [M+H]⁺, 320 (23) [M+NH₄]⁺, 325(24) [M+Na]⁺.

Example 60(E,E)-(1-Cyano-4-(3,4-dihydroxyphenyl)-buta-1,3-dienyl)phosphonic aciddiethyl ester

The compound was prepared as described in Example 3 by adding3,4-dihydroxycinnamaldehyde (32 mg, 0.2 mmol) to diethyl ester ofcyanomethylphosphonic acid (34 mg, 0.2 mmol). After refluxing for 3 hand recrystallization from ethanol-water an orange solid was obtained(45 mg, 70%). The product gave the following analytical data:

NMR (CD₃C(O)CD₃, δ, ppm): 1.35; 4.18 (2×m, 10H, P(OC₂H₅)₂, 6.91 (d, 1H,J=8.2 Hz, H⁵), 7.07 (ddd, 1H, J 1.9, 11.5 and 15.2 Hz, PhC═CH), 7.11(dd, 1H, J 2.1 and 8.2 Hz, H⁶), 7.24 (d, 1H, J=2.1 Hz, H²), 7.42 (d, 1H,J=15.2 Hz, PhCH═C), 7.80 and 7.84 (2×d, 2×0.5H, J=11.5 Hz, CH═CCN).

MS, m/e (rel. intensity, %): 324 (100) [M+H]⁺, 341 (62) [M+NH₄]⁺.

Example 61(E,E)-2-(3,4-Dimethoxybenzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile

To 3,4-dihydroxycinnamaldehyde (32 mg, 0.2 mmol) andN-(cyanoacetyl)3,4-dimethoxybenzylamide (47 mg, 0.2 mmol) in 8 mL ofethanol 40 μl of piperidine was added and the mixture was kept at roomtemperature for 1 h. 2N HCl and water were added and the precipitatedsolid was recrystallized from ethanol-water to give 52 mg (68%) of anorange solid. The product gave the following analytical data:

NMR (CD₃C(O)CD₃, δ, ppm): 3.79; 3.80 (2×s, 6H, Ph′(OCH₃)₂, 4.46 (s, 2H,CH₂Ph′), 6.89 (d, 1H, J=8.3 Hz, H⁵), 6.90; 7.01 (2×m, 2H, Ph′), 7.05(dd, 1H, J 11.7 and 15.2 Hz, PhC═CH), 7.07 (dd, 1H, J 2.1 and 8.3 Hz,H⁶), 7.21 (d, 1H, J=2.1 Hz, H²), 7.34 (d, 1H, J=15.2 Hz, PhCH═C), 8.00(d, 1H, J=11.7 Hz, CH═CCN).

MS, m/e (rel. intensity, %): 381 (100) [M+H]⁺, 398 (26) [M+NH₄]⁺.

Example 62(E,E)-2-(Benzylaminocarbonyl)-3-(4-hydroxy-3-methoxystyryl)acrylonitrile

The compound was prepared as described in Example 37 by addingpiperidine to a solution of 4-hydroxy-3-methoxycinnamaldehyde (35 mg,0.2 mmol) and N-(cyanoacetyl)benzylamide (34 mg, 0.2 mmol) in ethanol.After recrystallization from ethanol-water a yellow solid was obtained(50 mg, 75%). The product gave the following analytical data:

NMR (CD₃C(O)CD₃, δ, ppm): 3.92 (s, 3H, OMe), 4.55 (s, 2H, CH₂Ph′), 6.91(d, 1H, J=8.2 Hz, H⁵), 7.14 (dd, 1H, J 11.6 and 15.1 Hz, PhC═CH), 7.20(dd, 1H, J 2.1 and 8.2 Hz, H⁶), 7.25; 7.31-7.42 (2×m, 5H, Ph′), 7.35 (d,1H, J=15.1 Hz, PhCH═C), 7.37 (d, 1H, J=2.1 Hz, H²), 8.02 (d, 1H, J=11.6Hz, CH═CCN).

MS, m/e (rel. intensity, %): 335 (100) [M+H]⁺, 352 (30) [M+NH₄]⁺.

Example 63(E,E)-2-(3,4-Dihydroxybenzylaminocarbonyl)-3-(4-hydroxy-3-methoxystyryl)acrylonitrile

The compound was prepared as described in Example 37 by addingpiperidine to a solution of 4-hydroxy-3-methoxycinnamaldehyde (35 mg,0.2 mmol) and N-(cyanoacetyl)3,4-dihydroxybenzylamide (40 mg, 0.2 mmol)in ethanol. After recrystallization from ethanol-water a yellow solidwas obtained (53 mg, 72%). The product gave the following analyticaldata:

NMR (CD₃C(O)CD₃, δ, ppm): 3.92 (s, 3H, OMe), 4.38 (s, 2H, CH₂Ph′), 6.68(dd, 1H, J 2.1 and 8.1 Hz, H^(6′)), 6.76 (d, 1H, J 8.1 Hz, H^(5′)),6.86(d, 1H, J 2.1 Hz, H^(2′)), 6.91 (d, 1H, J=8.2 Hz, H′), 7.13 (dd, 1H,J 11.6 and 15.3 Hz, PhC═CH), 7.20 (dd, 1H, J 2.1 and 8.2 Hz, H⁶), 7.37(d, 1H, J=2.1 Hz, H²), 7.39 (d, 1H, J=15.3 Hz, PhCH═C), 8.00 (d, 1H,J=11.6 Hz, CH═CCN).

MS, m/e (rel. intensity, %): 367 (100) [M+H]⁺, 384 (45) [M+NH₄]⁺, 389(24) [M+Na]⁺.

Example 64(E,E)-2-((3-Trifluoromethyl)benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile

The compound was prepared as described in Example 37 by addingpiperidine to a solution of 3,4-dihydroxycinnamaldehyde (35 mg, 0.2mmol) and 2-cyano-N-(3-trifluoromethylbenzyl)acetamide (48 mg, 0.2 mmol)in ethanol. After recrystallization from ethanol-water a yellow solidwas obtained (54 mg, 70%). The product gave the following analyticaldata:

NMR (CD₃C(O)CD₃, δ, ppm): 4.65 (s, 2H, CH₂Ph′), 6.90 (d, 1H, J=8.2 Hz,H⁵), 7.05 (dd, 1H, J 11.6 and 15.1 Hz, PhC═CH), 7.08 (dd, 1H, J 2.2 and8.2 Hz, H⁶), 7.22 (d, 1H, J=2.2 Hz, H²), 7.36 (d, 1H, J=15.1 Hz,PhCH═C), 7.60; 7.70 (2×m, 2×2H, Ph′), 8.02 (d, 1H, J=11.6 Hz, CH═CCN).

MS, m/e (rel. intensity, %): 389 (100) [M+H]⁺, 406 (60) [M+NH₄]⁺.

Example 65(E,E)-2-(3-Fluorobenzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile

The compound was prepared as described in Example 37 by addingpiperidine to a solution of 3,4-dihydroxycinnamaldehyde (35 mg, 0.2mmol) and 2-cyano-N-(3-fluorobenzyl)acetamide (38 mg, 0.2 mmol) inethanol. After recrystallization from ethanol-water a yellow solid wasobtained (51 mg, 75%). The product gave the following analytical data:

NMR (CD₃C(O)CD₃, δ, ppm): 4.56 (s, 2H, CH₂Ph′), 6.90 (d, 1H, J=8.2 Hz,H⁵), 7.02; 7.14; 7.20; 7.36 (4×m, 4×1H, Ph′), 7.05 (dd, 1H, J 11.7 and15.3 Hz, PhC═CH), 7.07 (dd, 1H, J 2.2 and 8.2 Hz, H⁶), 7.22 (d, 1H,J=2.2 Hz, H²), 7.35 (d, 1H, J=15.3 Hz, PhCH═C), 8.01 (d, 1H, J=11.7 Hz,CH═CCN).

MS, m/e (rel. intensity, %): 339 (100) [M+H]⁺, 356 (12) [M+NH₄]⁺.

Example 66(E,E)-2-((Pyridin-4-ylmethyl)aminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile

The compound was prepared as described in Example 3 by adding3,4-dihydroxycinnamaldehyde (32 mg, 0.2 mmol) to2-cyano-N-pyridin-4-ylmethylacetamide (33 mg, 0.2 mmol). After refluxingfor 2 h and recrystallization from ethanol-water an orange solid wasobtained (44 mg, 69%). The product gave the following analytical data:

NMR (CD₃C(O)CD₃, δ, ppm): 4.59 (s, 2H, CH₂Ph′), 6.91 (d, 1H, J=8.2 Hz,H⁵), 7.07 (dd, 1H, J 11.7 and 15.3 Hz, PhC═CH), 7.09 (dd, 1H, J 2.2 and8.2 Hz, H⁶), 7.23 (d, 1H, J=2.2 Hz, H²), 7.37 (d, 1H, J=15.3 Hz,PhCH═C), 7.38 (m, 2H, H^(2′)+H^(6′)), 8.04 (d, 1H, J=11.7 Hz, CH═CCN),8.51 (m, 2H, H^(3′)+H^(5′)).

MS, m/e (rel. intensity, %): 284 (30), 322 (100) [M+H]⁺.

Example 67(E,E)-2-(3,4-Dihydroxybenzylaminocarbonyl)-3-(3-methoxy-4-hydroxy-5-nitrostyryl)acrylonitrile

The compound was prepared as described in Example 3 by adding3-methoxy-4-hydroxy-5-nitrocinnamaldehyde (22 mg, 0.1 mmol) toN-(cyanoacetyl)3,4-dihydroxybenzylamide (21 mg, 0.1 mmol). Afterrefluxing for 4 h and recrystallization from ethanol-water an orangesolid was obtained (19 mg, 46%). The product gave the followinganalytical data:

NMR (CD₃C(O)CD₃, δ, ppm): 4.02 (s, 3H, OMe), 4.38 (s, 2H, CH₂Ph′), 6.69(dd, 1H, J 2.1 and 8.0 Hz, H^(6′)), 6.76 (d, 1H, J=8.0 Hz, H^(5′)), 6.87(d, 1H, J=2.1 Hz, H^(2′)), 7.29 (dd, 1H, J 11.5 and 15.3 Hz, PhC═CH),7.47 (d, 1H, J=15.3 Hz, PhCH═C), 7.74 (d, 1H, J=1.9 Hz, H²), 7.90 (d,1H, J=1.9 Hz, H⁶), 8.02 (d, 1H, J=11.5 Hz, CH═CCN).

MS, m/e (rel. intensity, %): 293 (30), 412 (100) [M+H]⁺, 429 (35)[M+NH₄]⁺.

Example 68 3,4-Dihydroxycinnamaldehyde (A₁₂)

3,4-Bis(t-butyldimethylsilyloxy)cinnamyl alcohol A₉ (Example 10) (7.88g, 20 mmol) was dissolved in 1000 mL CH₂Cl₂, 17.2 g activated MnO₂ (200mmol) were added and the mixture was thoroughly stirred for 24 h at 20°C. MnO₂ was filtered off and the filtrate was taken to dryness. To theobtained 3,4-diBDMS caffeoyl aldehyde 250 mL of CHCl₃ was added followedby addition of n-Bu₄NF monohydrate (11.6 g, 40 mmol). The mixture wasstirred at 20° C. for 30 min and worked up with 300 mL of 5% HCl.Chloroform layer was separated, washed with H₂O, dried with Na₂SO₄ andtaken to dryness. The residue was purified on a silica gel column,eluent MeOH—CHCl₃, 1:4+1% AcOH. The solvents were evaporated and thecrystalline residue washed with CHCl₃ to give aldehyde A₁₂, yield 1.77 g(54%). The product gave the following analytical data:

NMR (CD₃C(O)CD₃, δ, ppm): 6.54 (dd, J 7.7, 15.8 Hz, Hα olefinic), 6.91(d, J=8.2 Hz, H⁵), 7.12 (br.d, J=8.2 Hz, H⁶), 7.21 (br.s, H²), 7.52 (d,J=15.8 Hz, Hβ olefinic), 9.62 (d, J=7.7 Hz, CHO).

Example 69(E,E)-2-(Benzylaminocarbonyl)-3-(3,4-dihydroxystyryl)acrylonitrile(CR4)—Method C

To 3,4-dihydroxycinnamaldehyde A₁₂ (example 59) (32 mg, 0.2 mmol) andamide A₃ (Example 4) (32 mg, 0.2 mmol) in 8 mL of ethanol 40 μl ofpiperidine was added and the mixture was kept at room temperature for 1h. 2N HCl and water were added and the precipitated solid wasrecrystallized from ethanol-water to give 44 mg (68%) of an orangesolid. The analytical data were identical to the compound prepared asdescribed in Example 8.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A compound of Formula I, or a salt, solvate, or hydrate thereof:

wherein R¹ and R² are each independently selected from H, OH, C₁₋₆alkyl,C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),C₁₋₆alkyl(C═O)NH, C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring; R³ isselected from H, OH, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylCO₂, NH₂,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), C₁₋₆alkyl(C═O)NH,C₁₋₆alkyl(C═O)N(C₁₋₆alkyl), SH, S—C₁₋₆alkyl,O—Si(C₁₋₆alkyl)(C₁₋₆alkyl)(C₁₋₆alkyl), NO₂, halo and CH₂—S—(CH₂), Ar; R⁴is selected from C(X)R⁵, SO₃Ar, NH₂, NH—C₁₋₆alkyl,N(C₁₋₆alkyl)(C₁₋₆alkyl), P(O)(OH)₂, P(O)(OC₁₋₆alkyl)₂, andC(NH₂)═C(CN)₂; X is selected from O, S, NH and N—C₁₋₆alkyl; R⁵ isselected from NH₂, OH, NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH,(CH₂)_(p)OC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkoxy, NHNH₂, NHC(O)NH₂,NHC(O)C₁₋₆alkoxy, N-morpholino and N-pyrrolidino; and Ar is an aromaticor heteroaromatic group, unsubstituted or substituted with 1-4substituents, independently selected from OH, C₁₋₆alkyl, C₁₋₆alkoxy,NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl), SH, S—C₁₋₆alkyl, NO₂, CF₃,OCF₃ and halo; n is 0 to 4; and p is 1-4.
 2. The compound according toclaim 1, wherein R¹ and R² are each independently selected from H, OH,C₁₋₄alkyl, C₁₋₄alkoxy, C₁₋₄alkylCO₂, NH₂, NH—C₁₋₄alkyl,C₁₋₄alkyl(C═O)NH, C₁₋₄alkyl(C═O)N(C₁₋₄alkyl), SH, S—C₁₋₄alkyl,O—Si(C₁₋₄alkyl)(C₁₋₄alkyl)(C₁₋₄alkyl), NO₂, CF₃, OCF₃ and halo, or R¹and R² together represent O—C₁₋₆alkyl-O, thereby forming a ring.
 3. Thecompound according to claim 2, wherein R¹ and R² are each independentlyselected from the group consisting H, OH, OCH₃, CH₃CO₂,O—Si(CH₃)₂(^(t)Bu), S-Me, SH, CH₃CONH, CH₃CONCH₃, and NO₂.
 4. Thecompound according to claim 3, wherein R¹ and R² are both OH or R¹ andR² are both OCH₃.
 5. The compound according to claim 4, wherein R¹ isOCH₃ and R² is OH.
 6. The compound according to claim 1, wherein R³ isselected from H, OH, C₁₋₄alkyl, C₁₋₄alkoxy, C₁₋₄alkylCO₂, NH₂,NH—C₁₋₄alkyl, N(C₁₋₄alkyl)(C₁₋₄alkyl), C₁₋₄alkyl(C═O)NH,C₁₋₄alkyl(C═O)N(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂ and halo.
 7. Thecompound according to claim 6, wherein R³ is selected from selected fromH, OH, OCH₃, CH₃CO₂, SH, SMe, NO₂, CH₃CONH, CH₃CONCH₃, and halo.
 8. Thecompound according to claim 1, wherein R¹, R², and R³ are eachindependently selected from H, C₁₋₄alkylCO₂, C₁₋₆alkyl(C═O)NH, andC₁₋₆alkyl(C═O)N(C₁₋₆alkyl), provided that at least one of R¹, R², and R³is not hydrogen.
 9. The compound according to claim 1, wherein R⁴ isselected from selected from C(X)R⁵ and C(NH₂)═C(CN)₂.
 10. The compoundaccording to claim 9, wherein R⁴ is C(X)R⁵.
 11. The compound accordingto claim 10, wherein X is selected from selected from O and S.
 12. Thecompound according to claim 10, wherein R⁵ is selected from selectedfrom NH₂, OH, NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH and C₁₋₄alkoxy.
 13. Thecompound according to claim 12, wherein p is 1-3.
 14. The compoundaccording to claim 13, wherein R⁵ is selected from selected from NH₂,OH, NH(CH₂)_(p)Ar, NH(CH₂)_(p)OH and OCH₃.
 15. The compound according toclam 14, wherein p is 1-2.
 16. The compound according to claim 1,wherein Ar is an unsubstituted phenyl group or a phenyl groupsubstituted with 1-4 substituents optionally selected from selected fromOH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁ alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo.
 17. The compound according toclaim 14, wherein Ar is an unsubstituted phenyl group or a phenyl groupsubstituted with 1-4 substituents optionally selected from selected fromOH, C₁₋₆alkyl, C₁₋₆alkoxy, NH₂, NH—C₁₋₆alkyl, N(C₁₋₆alkyl)(C₁₋₆alkyl),SH, S—C₁₋₆alkyl, NO₂, CF₃, OCF₃ and halo.
 18. The compound according toany of claims 16 and 17, wherein Ar is an unsubstituted phenyl group orphenyl group substituted with 1-2 substituents optionally selected fromselected from OH, C₁₋₄alkyl, C₁₋₄alkoxy, NH₂, NH—C₁₋₄alkyl,N(C₁₋₄alkyl)(C₁₋₄alkyl), SH, S—C₁₋₄alkyl, NO₂, CF₃, OCF₃ and halo. 19.The compound according to claim 18, wherein Ar is an unsubstitutedphenyl group or phenyl group substituted with 1-2 substituentsoptionally selected from OH, OCH₃, NH₂, NHCH₃, N(CH₃)₂, SH, SCH₃, CF₃,OCF₃ and halo.
 20. A compound selected from:


21. A composition comprising a compound according to any one of claims 1to 20 in admixture with a pharmaceutically acceptable diluent orcarrier.
 22. A use of a compound capable of modulating cellproliferation according to any one of claims 1 to 20 to prepare amedicament to modulate cell proliferation.
 23. A use of a compoundcapable of inhibiting cell proliferation according to any one of claims1 to 20 to inhibit cell proliferation.
 24. A use of a compound capableof inhibiting cancer cell proliferation according to any one of claims 1to 20 to inhibit cancer cell proliferation.
 25. A use of a compoundaccording to any one of claims 1 to 20 to treat cancer.
 26. A useaccording to claim 24 or 25 wherein said cancer is a hematopoietic cellcancer.
 27. A use according to claim 24 or 25 wherein said cancer is aleukemia, a lymphoma, a myeloma or a carcinoma.
 28. A use according toclaim 27 wherein said leukemia is acute lymphoblastic leukemia,Philadelphia+ leukemia, Philadelphia− leukemia, acute myelocyticleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia orjuvenile myelomonocyte leukemia.
 29. A use according to claim 27 whereinsaid leukemia is acute lymphoblastic leukemia.
 30. A method ofmodulating cell proliferation comprising administering an effectiveamount of a compound capable of modulating cell proliferation accordingto any one of claims 1 to 20 or a composition according to claim 21 to acell or animal in need thereof.
 31. A method of inhibiting cellproliferation comprising administering an effective amount of a compoundcapable of inhibiting cell proliferation according to any one of claims1 to 20 or a composition according to claim 21 to a cell or animal inneed thereof.
 32. A method of inhibiting cancer cell proliferationcomprising administering an effective amount of a compound capable ofinhibiting cancer cell proliferation according to any one of claims 1 to20 or a composition according to claim 21 to a cell or animal in needthereof.
 33. A method of treating cancer comprising administering aneffective amount of a compound capable of inhibiting cancer cellproliferation according to any one of claims 1 to 20 or a compositionaccording to claim 21 to a cell or animal in need thereof.
 34. A methodaccording to claim 32 or 33 wherein said cancer is a hematopoietic cellcancer.
 35. A method according to claim 32 or 33 wherein said cancer isa leukemia, a lymphoma, a myeloma or a carcinoma.
 36. A method accordingto claim 35 wherein said leukemia is acute lymphoblastic leukemia,aggressive Philadelphia+ leukemia, acute myelocytic leukemia, chronicmyeloid leukemia, chronic lymphocytic leukemia or juvenile myelomonocyteleukemia,
 37. A method according to claim 35 wherein said leukemia isacute lymphoblastic leukemia.